On-chip microfluidic processing of particles

ABSTRACT

A microfluidic device comprises: a channel extending from a plurality of inlets to a plurality of outlets, wherein the channel is bounded by a first wall and a second wall opposite from the first wall; and an array of obstacles disposed within the channel configured to deflect particles in a sample comprising the particles toward the second wall when the particles are flowed from the inlets to the outlets. The particles are inputted into at least one of the plurality of inlets and are deflected through a series of parallel flow streams flowing from the plurality of inlets to the plurality of outlets while being deflected toward the second wall, wherein streams in the parallel flows comprise a reagent. Microfluidic devices and methods greatly improve cell quality, streamline workflows, and lower costs. Applications include research and clinical diagnostics in cancer, infectious disease, and inflammatory disease, among other disease areas.

CROSS-REFERENCE

The application claims the benefit of U.S. Provisional PatentApplication No. 61/939,044, filed on Feb. 12, 2014, U.S. ProvisionalPatent Application No. 61/939,070, filed on Feb. 12, 2014, and U.S.Provisional Patent Application No. 61/800,222, filed on Mar. 15, 2013,which applications are herein incorporated by reference in theirentireties.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with the support of the United States governmentunder Contract number 1R41CA174121-01 by the National Institutes ofHealth (NIH).

BACKGROUND

Current methods for sample preparation of leukocytes prior tomulti-parameter analysis via flow cytometry involve centrifugation andare tedious, manual processes that require expert operators and resultin lost and damaged cells. Multi-parameter flow cytometry or atomic massspectrometry is an increasingly powerful and widely used technology inresearch and clinical diagnostic testing for cancer, infectiousdiseases, inflammatory diseases, and many other diseases. However,membrane and intracellular labeling of cells for multi-parameter flowcytometry can be a labor- and time-intensive process that can result ina significant loss of cells. Since each centrifugal wash/concentratestep in the conventional particle (e.g., cell)-processing process canhave a cell yield of only ˜80-90% and since multiple washes can berequired for each of the steps, the overall process can take severalhours and overall cell yield can be <50%. Low cell yield can necessitatelarger blood samples, an especially critical problem for small childrenand for patients with anemia or who need many blood tests. Animalstudies, too, can be hampered by the limited sample volumes available,e.g. from mice. Because these steps are done by hand, the results can behighly variable. Thus, there is a need for improved methods for chemicaland/or enzymatic treatment, washing, and isolation of particles (e.g.,cells) obtained from samples comprising particles (e.g., cells), and useof microfluidic devices can enhance these particle (e.g.,cell)-processing procedures.

SUMMARY

In one aspect, described herein are microfluidic devices and methodsthat can greatly improve cell quality and quantity, streamlineworkflows, and lower costs. Applications can include research andclinical diagnostics in cancer, infectious disease, and inflammatorydisease, among other disease areas. These devices and methods canfulfill a significant unmet need in both research and clinical settingsfor high leukocyte recovery and quick sample processing, leading tohigher quality results and cost/efficiency gains.

In one aspect, provided herein is a device comprising: a.) a channelextending from a plurality of inlets to a plurality of outlets, whereinthe channel is bounded by a first wall and a second wall opposite fromthe first wall; and b.) an array of obstacles disposed within thechannel configured to deflect particles in a sample comprising theparticles toward the second wall when the particles are flowed from theinlets to the outlets, wherein the device is configured such that theparticles are inputted into at least one of the plurality of inlets andare deflected through a series of parallel flow streams flowing from theplurality of inlets to the plurality of outlets while being deflectedtoward the second wall, wherein at least four flow streams in the seriesof parallel flow streams comprise a reagent. In some cases, the deviceis microfluidic. In some cases, the particles are cells. In some cases,the cells are leukocytes or a subtype of leukocytes. In some cases, thecells are stem cells, cancer cells, or leukemia cells. In some cases,the stem cells are derived from umbilical cord blood. In some cases, thesurface of the device is hydrophilic. In some cases, the obstacles aremade from a polymer. In some cases, the device further comprises aplurality of reservoirs in fluid communication with the inlets. In somecases, the plurality of reservoirs comprise a sample, a buffer, a cellsurface label, a fix and permeabilize reagent, an intracellular label,or any combination thereof. In some cases, the device further comprisesan analytical device in fluid communication with at least one of aplurality of outlets, wherein the analytical device is configured toperform an analysis of particles processed by the device. In some cases,the analytical device comprises a flow cytometer or a mass spectrometer.In some cases, at least one of the flow streams comprises a bindingagent, wherein the binding agent comprises a label, wherein the label isdetectable. In some cases, the binding agent is an antibody. In somecases, the label is a fluorophore. In some cases, at least one of theflow streams comprises a fixation reagent. In some cases, at least oneof the flow streams comprises a permeabilization reagent. In some cases,the series of parallel flow streams comprises a first reagent flowstream comprising a first binding agent, a second reagent flow streamcomprising a fixation agent, a third reagent flow stream comprising apermeabilization reagent, and a fourth reagent flow stream comprising asecond binding agent, wherein the first binding agent comprises a firstdetectable label, and wherein the second binding agent comprises asecond detectable label. In some cases, the first and second detectablelabel comprise different labels. In some cases, the first and/or secondbinding agent is an antibody. In some cases, the label is a fluorophore.In some cases, the particles deflected toward the second wall flowthrough the first reagent flow stream, followed by the second reagentflow stream, followed by the third reagent flow stream, followed by thefourth reagent flow stream. In some cases, each of the reagent flowstreams is separated by a wash stream comprising a wash buffer. In somecases, the channel is from about 0.15 cm to about 20 cm long. In somecases, the channel is from about 0.05 mm to about 5 mm wide. In somecases, each of the parallel flow streams is from about 50 to 500 μmwide. In some cases, the particles deflected toward the second wallcomprise particles of a predetermined size. In some cases, the particlesof a predetermined size comprise particles above a critical size betweenobstacles within the array of obstacles. In some cases, the criticalsize is about 5 μm. In some cases, the array of obstacles extends acrossthe channel. In some cases, the obstacles are arranged in rows andcolumns, wherein the rows define an array direction that differs fromthe flow of the plurality of parallel flow streams by a tilt angle (ε)that has a magnitude greater than zero and less than or equal to ⅓radian, the obstacles in each respective column defining gaps betweenthe obstacles through which the fluid flows generally transversely withrespect to the columns, and wherein the obstacles are shaped such thatsurfaces of two obstacles defining a respective gap are asymmetricallyoriented about a first plane that extends through the center of therespective gap and that is parallel to the flow of the plurality ofparallel flow streams. In some cases, the columns repeat periodically.In some cases, the columns have a periodicity that repeats and is equalto 1/ε, wherein ε is measured in radians. In some cases, the rows andcolumns are at an angle of 90 degrees with respect to one another. Insome cases, each of the two obstacles defining a respective gap has acircular cross-section. In some cases, the obstacles have a diameter of18 μm. In some cases, a gap between each of the obstacles in both thehorizontal and vertical direction is 18 μm. In some cases, the tiltangle is 1/42 radians. In some cases, each of the two obstacles defininga respective gap has a triangular cross-section. In some cases, thetriangular cross-section comprises an isosceles triangle, comprising twoequal sides and one non-equal side. In some cases, the non-equal side isoriented parallel to flow of the plurality of parallel flow streams, andwherein the vertex opposite the non-equal side points toward the secondwall. In some cases, the gap from the vertex opposite the non-equal sideof one of the two obstacles defining a respective gap to the non-equalside of the other of the two obstacles defining a respective gap is 26μm. In some cases, a distance from the middle of the non-equal sideoriented parallel to the flow of the plurality of parallel flow streamsto the vertex opposite the middle is about 52 μm, and wherein the lengthof the non-equal side is about 52 μm. In some cases, the columns have aperiod of 76 μm. In some cases, the tilt angle is 1/36 radians. In somecases, the array of obstacles comprises at least one separator walloriented parallel to flow of the plurality of flow streams and the firstand second walls, wherein the particles introduced into an sample inletnear the first wall pass through the plurality of flow streams whilebeing deflected toward the second wall. In some cases, the at least oneseparator wall is configured to delay the flow of deflected particlestoward the second wall, wherein the delay serves to substantiallyincrease an amount of time that deflected particles reside in a flowstream, and/or substantially reduce mixing between parallel flowstreams. In some cases, the sample comprises EDTA. In some cases, theconcentration of EDTA in the sample is at least 5 mM. In some cases, thesample comprises acid citrate dextrose. In some cases, the samplecomprises a thrombin inhibitor. In some cases, the thrombin inhibitor isPPACK. In some cases, the concentration of PPACK in the sample is atleast 40 μM. In some cases, the sample comprises an agent that reducesthe activity of calcium-dependent integrins or an agent that reducescalcium dependent thrombin formation and a thrombin inhibitor.

In one aspect, provided herein is a device comprising: a.) a channelextending from a plurality of inlets to a plurality of outlets, whereinthe channel is bounded by a first wall and a second wall opposite fromthe first wall, and wherein the device is configured to flow a pluralityof flow streams from the plurality of inlets to the plurality ofoutlets, wherein the plurality of flow streams flow parallel to eachother; and b.) an array of obstacles disposed within the channelconfigured to deflect particles in a sample comprising the particlestoward the second wall when the particles are flowed from the inlets tothe outlets, wherein the array of obstacles comprises at least oneseparator wall oriented parallel to flow of the plurality of flowstreams and the first and second walls, wherein particles introducedinto an sample inlet near the first wall pass through the plurality offlow streams while being deflected toward the second wall, and whereinthe at least one separator wall is configured to delay the flow ofdeflected particles toward the second wall, wherein the delay serves tosubstantially increase an amount of time that deflected particles residein a flow stream, and/or substantially reduce mixing between parallelflow streams. In some cases, the array of obstacles comprises a seriesof separator walls wherein all separator walls in the series ofseparator walls extends from either an inlet or outlet portion of thechannel, and wherein each of the separator walls in the series ofseparator walls extends further into the array obstacles than theprevious separator wall. In some cases, the array of obstacles comprisesat least one pair of opposing separator walls, wherein the pair ofseparator walls comprises a first separator wall that extends from aninlet portion of the channel, and a second separator wall that extendsfrom an outlet portion of the channel, wherein the first and secondseparator walls are configured to substantially limit mixing betweenadjacent parallel flow streams, and wherein a gap exists between thepair of opposing separating walls, wherein the gap is configured toallow particles deflected through the adjacent parallel flow streamstoward the second wall to pass between the pair of opposing separatorwalls and continue flowing through the array of obstacles. In somecases, each separator wall has a variable width along the length of theseparator wall. In some cases, the device is microfluidic. In somecases, the particles are cells. In some cases, the cells are leukocytes.In some cases, the cells are stem cells. In some cases, the stem cellsare derived from umbilical cord blood. In some cases, the surface of thedevice is hydrophilic. In some cases, the obstacles are made from apolymer. In some cases, the device further comprises a plurality ofreservoirs in fluid communication with the inlets. In some cases, theplurality of reservoirs comprise a sample, a buffer, a cell surfacebinding agent comprising a first label, a fixation and permeabilizationreagent, an intracellular binding agent comprising a second label, orany combination thereof. In some cases, the first and/or second label isdetectable. In some cases, the first and second label are different. Insome cases, the first and/or second label is a fluorophore. In somecases, at least one of a plurality of outlets is fluidly coupled to ananalytical device, wherein the analytical device is configured toperform an analysis of particles processed by the device. In some cases,the analytical device comprises a flow cytometer or a mass spectrometer.In some cases, at least one of the flow streams comprises a bindingagent, wherein the binding agent comprises a label, wherein the label isdetectable. In some cases, the binding agent is an antibody. In somecases, the label is a fluorophore. In some cases, at least one of theflow streams comprises a fixation reagent. In some cases, at least oneof the flow streams comprises a permeabilization reagent. In some cases,the plurality of flow streams is arranged in a series of reagent flowstreams wherein a first of the plurality of flow streams comprises afirst binding agent comprising a first label, a second of the pluralityof flow streams comprises a fixation and permeabilization agent, and athird of the plurality of flow streams comprises a second binding agentcomprising a second label. In some cases, the second of the plurality offlow streams is split into one reagent flow stream comprising a fixationagent and another reagent flow stream comprising a permeabilizationagent. In some cases, the particles deflected toward the second wallflow through the first reagent flow stream, followed by the secondreagent flow stream, followed by the third reagent flow stream. In somecases, each of the reagent flow streams is separated by a wash streamcomprising a wash buffer. In some cases, the first and/or second labelis detectable. In some cases, the first and second label are different.In some cases, the first and/or second label is a fluorophore. In somecases, the channel is from about 0.15 cm to about 20 cm long. In somecases, the channel is from about 0.05 mm to about 5 mm wide. In somecases, each of the plurality of flow streams is from about 50 to 500 μmwide. In some cases, the particles deflected toward the second wallcomprise particles of a predetermined size. In some cases, the particlesof a predetermined size comprise particles above a critical size betweenobstacles within the array of obstacles. In some cases, the criticalsize is about 5 μm. In some cases, the array of obstacles extends acrossthe channel. In some cases, the obstacles are arranged in rows andcolumns, wherein the rows define an array direction that differs fromthe flow of the plurality of parallel flow streams by a tilt angle (ε)that has a magnitude greater than zero and less than or equal to ⅓radian, the obstacles in each respective column defining gaps betweenthe obstacles through which the fluid flows generally transversely withrespect to the columns, and wherein the obstacles are shaped such thatsurfaces of two obstacles defining a respective gap are asymmetricallyoriented about a first plane that extends through the center of therespective gap and that is parallel to the flow of the plurality ofparallel flow streams. In some cases, the columns repeat periodically.In some cases, the columns have a periodicity that repeats and is equalto 1/ε, wherein ε is measured in radians. In some cases, the rows andcolumns are at an angle of 90 degrees with respect to one another. Insome cases, each of the two obstacles defining a respective gap has acircular cross-section. In some cases, the obstacles have a diameter of18 μm. In some cases, a gap between each of the obstacles in both thehorizontal and vertical direction is 18 μm. In some cases, the tiltangle is 1/42 radians. In some cases, each of the two obstacles defininga respective gap has a triangular cross-section. In some cases, thetriangular cross-section comprises an isosceles triangle, comprising twoequal sides and one non-equal side. In some cases, the non-equal side isoriented parallel to flow of the plurality of parallel flow streams, andwherein the vertex opposite the non-equal side points toward the secondwall. In some cases, the gap from the vertex opposite the non-equal sideof one of the two obstacles defining a respective gap to the non-equalside of the other of the two obstacles defining a respective gap is 26μm. In some cases, a distance from the middle of the non-equal sideoriented parallel to the flow of the plurality of parallel flow streamsto the vertex opposite the middle is about 52 μm, and wherein the lengthof the non-equal side is about 52 μm. In some cases, the columns have aperiod of 76 μm. In some cases, the tilt angle is 1/36 radians. In somecases, the sample comprises EDTA. In some cases, the concentration ofEDTA in the sample is at least 5 mM. In some cases, the sample comprisesacid citrate dextrose. In some cases, the sample comprises a thrombininhibitor. In some cases, the thrombin inhibitor is PPACK. In somecases, the concentration of PPACK in the sample is at least 40 μM. Insome cases, the sample comprises an agent that reduces the activity ofcalcium-dependent integrins or an agent that reduces calcium dependentthrombin formation and a thrombin inhibitor.

In one aspect, provided herein is a device comprising: a.) a channelextending from at least one inlet to a plurality of outlets, wherein thechannel is bounded by a first wall and a second wall opposite from thefirst wall; and b.) an array of obstacles disposed within the channelconfigured to deflect particles in a sample comprising the particlestoward the second wall when a stream comprising the particles is flowedfrom the at least one inlet to the plurality of outlets, wherein thefirst wall comprises a plurality of inlets adapted to flow a fluidtowards a plurality of outlets in the second wall, wherein the directionof the flow of the fluid is perpendicular to flow of the streamcomprising the particles and wherein the flow of the fluid is configuredto remove any particles that have become clogged in the array ofobstacles following movement of the particles toward the second wall. Insome cases, the channel further comprises a first pair of removablebarriers, wherein the first pair of removable barriers is configured toblock the at least one inlet and the plurality of outlets in the channelwhen the fluid is flowed from the first wall comprising a plurality ofinlets towards the plurality of outlets in the second wall. In somecases, the channel further comprises a second pair of removablebarriers, wherein the second pair of removable barriers is configured toblock the first wall comprising a plurality of inlets and the pluralityof outlets in the second wall when the stream comprising particles isflowed from the at least one inlet to the plurality of outlets. In somecases, the channel comprises a plurality of inlets, wherein the deviceis configured to flow a plurality of flow streams from the plurality ofinlets to the plurality of outlets, wherein the plurality of flowstreams flow parallel to each other. In some cases, the plurality offlow streams is arranged in a series of reagent flow streams. In somecases, the series comprises a first flow stream comprising a firstlabeling reagent, a second flow stream comprising a fixation andpermeabilization agent, and a third flow streams comprising a secondlabeling reagent. In some cases, the second flow stream is split intoone reagent flow stream comprising a fixation agent and another reagentflow stream comprising a permeabilization agent. In some cases, theparticles deflected toward the second wall flow through the series ofreagent flow streams. In some cases, each of the reagent flow streams isseparated by a wash stream comprising a wash buffer. In some cases, thearray of obstacles further comprises at least one separator walloriented parallel to the flow of the plurality of flow streams. In somecases, the at least one separator wall is configured to be removed fromthe array of obstacles when the fluid is flowed from the first wallcomprising a plurality of inlets towards the plurality of outlets in thesecond wall. In some cases, the sample comprises EDTA. In some cases,the concentration of EDTA in the sample is at least 5 mM. In some cases,the sample comprises acid citrate dextrose. In some cases, the samplecomprises a thrombin inhibitor. In some cases, the thrombin inhibitor isPPACK. In some cases, the concentration of PPACK in the sample is atleast 40 μM. In some cases, the sample comprises an agent that reducesthe activity of calcium-dependent integrins or an agent that reducescalcium dependent thrombin formation and a thrombin inhibitor.

In one aspect, provided herein is a method for labeling cells, themethod comprising: a.) providing a sample comprising cells; b.)processing the sample comprising cells, wherein the processing comprisesintroducing the sample comprising cells into a sample inlet of a devicecomprising an array of obstacles, and passing the sample through thearray of obstacles, wherein the array of obstacles comprises a pluralityof parallel flow streams flowing through the array of obstacles, whereinthe passing comprises flowing cells from the sample from the sampleinlet through the plurality of parallel flow streams, wherein at leastone of the plurality of parallel flow streams comprises a labelingreagent, at least one of the plurality of parallel flow streamscomprises a fixation agent, at least one of the plurality of parallelflow streams comprises a permeabilization agent, and at least one of theplurality of parallel flow streams comprises a wash buffer, whereby thepassing the sample through the array of obstacles serves to label thecells while also simultaneously separating the cells by size; and c.)harvesting labeled cells of a predetermined size from one of a pluralityof outlets of the device. In some cases, the labeling reagent comprisesa binding agent that comprises a label, wherein the label is detectable.In some cases, the label comprises a fluorophore. In some cases, thecells are leukocytes. In some cases, the cells are stem cells. In somecases, the stem cells are derived from umbilical cord blood. In somecases, the sample comprises sub-populations of different types ofleukocytes (granulocytes, lymphocytes, monocytes), and wherein relativeratios of the sub-populations are not substantially skewed. In somecases, erythrocytes are not lysed. In some cases, the sample comprisingcells is blood. In some cases, the blood is umbilical cord blood. Insome cases, clogging of the cells flowing through the array of obstaclesis substantially reduced by adding a solution comprising a calciumchelator and/or thrombin inhibitor to the sample prior to and concurrentwith flowing the sample through the device. In some cases, the calciumchelator is EDTA, wherein EDTA is added to a concentration of about 5mM. In some cases, the calcium chelator is acid citrate dextrose (ACD),wherein ACD is added to a concentration of about 10% v/v. In some cases,the thrombin inhibitor is (PPACK), wherein PPACK is added to aconcentration of about 40 μM. In some cases, addition of the solution tothe sample produces about a 40-fold reduction is clogging. In somecases, the yield of labeled cells is at least 85%. In some cases, theviability of the labeled cells is at least 90%. In some cases,centrifugation is not used. In some cases, erythrocytes are not lysed.In some cases, the method is performed in less than one hour. In somecases, the sample has a volume of less than 300 mL. In some cases, thedevice comprises a channel extending from the sample inlet to theplurality of outlets, wherein the channel is bounded by a first wall anda second wall opposite from the first wall, and wherein the array ofobstacles is disposed within the channel. In some cases, the devicecomprises a plurality of inlets, wherein one of the plurality of inletscomprises the sample inlet, while each of the plurality of parallel flowstreams flows through separate one of the plurality of inlets. In somecases, the device is microfluidic. In some cases, the surface of thedevice is hydrophilic. In some cases, the obstacles are made from apolymer. In some cases, the method further comprises a plurality ofreservoirs in fluid communication with the device. In some cases, theplurality of reservoirs comprise a sample, a buffer, a cell surfacelabel, a fix and permeabilize reagent, an intracellular label, or anycombination thereof. In some cases, the method further comprisesinputting a product from at least one of a plurality of outlets to ananalytical device in fluid communication with the at least one of theplurality of outlets, wherein the analytical device is configured toperform an analysis of particles processed by the device. In some cases,the analytical device comprises a flow cytometer or a mass spectrometer.In some cases, the plurality of parallel flow streams are arranged in asuccessive series of parallel reagent flow streams, wherein a firstreagent flow stream comprises a first binding agent comprising a firstlabel, a second reagent flow stream comprises a fixation agent, a thirdreagent flow stream comprises a permeabilization reagent, and a fourthreagent flow stream comprises a second binding agent comprising a secondlabel. In some cases, each of the reagent flow streams is separated by awash stream comprising a wash buffer. In some cases, the channel is fromabout 0.15 cm to about 20 cm long. In some cases, the channel is fromabout 0.05 mm to about 5 mm wide. In some cases, each of the parallelflow streams is from about 50 to 500 μm wide. In some cases, theparticles of a predetermined size comprise particles above a criticalsize between obstacles within the array of obstacles. In some cases, thecritical size is about 5 μm. In some cases, the array of obstaclesextends across the channel. In some cases, the obstacles are arranged inrows and columns, wherein the rows define an array direction thatdiffers from the flow of the plurality of parallel flow streams by atilt angle (ε) that has a magnitude greater than zero and less than orequal to ⅓ radian, the obstacles in each respective column defining gapsbetween the obstacles through which the fluid flows generallytransversely with respect to the columns, and wherein the obstacles areshaped such that surfaces of two obstacles defining a respective gap areasymmetrically oriented about a first plane that extends through thecenter of the respective gap and that is parallel to the flow of theplurality of parallel flow streams. In some cases, the columns repeatperiodically. In some cases, the columns have a periodicity that repeatsand is equal to 1/ε, wherein ε is measured in radians. In some cases,the rows and columns are at an angle of 90 degrees with respect to oneanother. In some cases, each of the two obstacles defining a respectivegap has a circular cross-section. In some cases, the obstacles have adiameter of 18 μm. In some cases, a gap between each of the obstacles inboth the horizontal and vertical direction is 18 μm. In some cases, thetilt angle is 1/42 radians. In some cases, each of the two obstaclesdefining a respective gap has a triangular cross-section. In some cases,the triangular cross-section comprises an isosceles triangle, comprisingtwo equal sides and one non-equal side. In some cases, the non-equalside is oriented parallel to flow of the plurality of parallel flowstreams, and wherein the vertex opposite the non-equal side pointstoward the second wall. In some cases, the gap from the vertex oppositethe non-equal side of one of the two obstacles defining a respective gapto the non-equal side of the other of the two obstacles defining arespective gap is 26 μm. In some cases, a distance from the middle ofthe non-equal side oriented parallel to the flow of the plurality ofparallel flow streams to the vertex opposite the middle is about 52 μm,and wherein the length of the non-equal side is about 52 μm. In somecases, the columns have a period of 76 μm. In some cases, the tilt angleis 1/36 radians. In some cases, the array of obstacles further comprisesat least one separator wall oriented parallel to the flow of theplurality of flow streams, wherein the at least one separator wall isconfigured to delay the flow of cells through the array of obstacles,wherein the delay serves to substantially increase an amount of timethat cells reside in a flow stream, and/or substantially reduce mixingbetween parallel flow streams. In some cases, the first wall furthercomprises a plurality of inlets adapted to flow a fluid towards aplurality of outlets in the second wall, wherein the direction of theflow of the fluid is perpendicular to flow of the sample comprising thecells and wherein the flow of the fluid is configured to remove anycells that have become clogged in the array of obstacles following flowof the cells through the array of obstacles. In some cases, the at leastone separator wall is configured to be removed from the array ofobstacles when the fluid is flowed from the first wall comprising aplurality of inlets towards the plurality of outlets in the second wall.In one aspect, provided herein is a method for processing leukocytes formolecular diagnostic testing, the method comprising labeling andharvesting the leukocytes from a sample using a microfluidic device,wherein the yield of labeled cells is at least 85% and the viability ofthe labeled cells is at least 90%. In some cases, the sample comprisessub-populations of different types of leukocytes (granulocytes,lymphocytes, monocytes), and wherein relative ratios of thesub-populations are not substantially skewed. In some cases,centrifugation is not used. In some cases, erythrocytes are not lysed.In some cases, the method is performed in less than one hour. In somecases, the sample has a volume of less than 300 mL. In some cases, thesample comprising cells is blood. In some cases, the blood is umbilicalcord blood. In some cases, clogging of the leukocytes flowing throughthe microfluidic device is substantially reduced by adding a solutioncomprising a calcium chelator and/or thrombin inhibitor to the sampleprior to and concurrent with labeling and harvesting the sample throughthe microfluidic device. In some cases, the calcium chelator is EDTA,wherein EDTA is added to a concentration of about 5 mM. In some cases,the calcium chelator is acid citrate dextrose (ACD), wherein ACD isadded to a concentration of about 10% v/v. In some cases, the thrombininhibitor is (PPACK), wherein PPACK is added to a concentration of about40 μM. In some cases, addition of the solution to the sample producesabout a 40-fold reduction is clogging.

In one aspect, provided herein is a system for processing and analyzingparticles, the system comprising: a.) a plurality of reservoirs, whereinat least one of the reservoirs comprises a sample comprising particles,and at least one of the reservoirs comprises a reagent; b.) a device,wherein the device is in fluid communication with each of the pluralityof reservoirs, and wherein the device is adapted to process particlesfrom the sample comprising particles, wherein the processing comprisesflowing the sample comprising particles from the reservoir comprisingthe sample into an input of a device, and passing the particles throughthe device, wherein the passing comprises flowing the particles from theinput through a plurality of parallel flow streams within the device,wherein at least one of the parallel flow streams comprises a reagentwhich flows from at least one of the plurality of reservoirs, andwherein the device comprises an array of obstacles, whereby the passingthe particles through the device serves to process the particles as wellas separate the particles by size; and c.) an analytical device in fluidcommunication with at least one of a plurality of outlet ports of thedevice, wherein the analytical device is configured to perform ananalysis of particles processed by the device.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 is a schematic diagram of cross-section of a “bump array” devicehaving right triangularly-shaped obstacles disposed in a microfluidicchannel. In the figure, fluid flow alternates between the right-to-leftand left-to-right directions, as indicated by the double-headed arrowmarked, “Fluid Flow.” In this array, right triangular posts are disposedin a square lattice arrangement that is tilted with respect directionsof fluid flow. The tilt angle ε (epsilon) is chosen so the device isperiodic. In this embodiment, a tilt angle of 18.4 degrees (⅓ radian)makes the device periodic after three rows. The gap between posts isdenoted G with triangle side length S and array pitch P. Streamlines areshown extending between the posts, dividing the fluid flow between theposts into three regions (“stream tubes”) of equal volumetric flow.

FIGS. 2A, 2B, and 2C, shows the trajectories of spherical polystyrenebeads of three different sizes in an array of the type shown in FIG. 1as the direction of fluid flow is cycled back and forth twice. Theorientation of the right triangular posts is denoted in the lower rightof each figure. Right isosceles triangles are 6 microns on a side withpost to post separation of 10 microns and a tilt angle of 5.71 degrees(0.1 radian). Particle sizes are 1.1 microns in FIG. 2A, 3.1 microns inFIG. 2B, and 1.9 microns in FIG. 2C. Particles shown in FIGS. 2A and 2Bretrace their paths when the direction of the fluid is switched, withthe particles in FIG. 2A generally following the fluid direction in eachfluid flow direction and the particles in FIG. 2B generally followingthe array direction in each fluid flow direction. By contrast, thetrajectory of the particles shown in FIG. 2C varies with the directionof the fluid flow. In FIG. 2C, small arrows indicate the direction ofthe fluid along the particle path; the particles generally follow thefluid direction when the fluid flow direction is left-to-right andgenerally follow the array direction when the fluid flow direction isright-to-left.

FIG. 3A shows simulated trajectories of 1.0-micrometer diameterparticles moving through an array of right triangular posts disposed ina microfluidic flow channel in which fluid flow alternates between theright-to-left and left-to-right directions. FIG. 3B shows simulatedtrajectories of 3.6-micrometer diameter particles moving through anarray of right triangular posts disposed in a microfluidic flow channelin which fluid flow alternates between the right-to-left and leftto-right directions. FIG. 3C shows simulated trajectories of3.2-micrometer diameter particles moving through an array of righttriangular posts disposed in a microfluidic flow channel in which fluidflow alternates between the right-to-left and left to-right directions.In these diagrams, the 1.0-micrometer diameter particles are smallerthan the critical size of the array in both fluid flow directions, the3.6-micrometer diameter particles are larger than the critical size ofthe array in both fluid flow directions, and the 3.2-micrometer diameterparticles are smaller than the critical size of the array in one(right-to-left) flow direction, but larger than the critical size of thearray in the other (left-to-right) flow direction.

FIG. 4A is a graph showing simulated normalized velocity flow betweentwo right triangular posts.

FIG. 4B is a graph showing normalized velocity profiles through gapsbetween round obstacles (curve that is symmetrical about Y/Gap=0.5) andright triangularly-shaped obstacles in an array of the type shown inFIG. 1 (ε=⅓ radian). In these profiles, vertical lines delineate theareas under each curve into thirds, representing three stream tubes ofequal volumetric flow. The curve for the round obstacles demonstratesthat one third of the volumetric flow between round obstacles occurs ina stream tube that is adjacent to either obstacle and has a width thatis 38% of the gap width. The curve for the triangular obstaclesdemonstrates that one third of the volumetric flow between triangularobstacles occurs in a stream tube that is adjacent to the flat side ofone of the two triangular obstacles and has a width that is 42% of thegap width and that an additional one third occurs in a stream tube thatis adjacent to the sharp side of the pair of triangular obstacles andhas a width that is 34% of the gap width.

FIG. 5 is a graph of predicted critical diameter versus the array tiltangle (ε) for arrays of triangular (lower line) and circular (upperline) obstacles.

FIG. 6A is a schematic diagram of cross-section of a “bump array” devicehaving equilateral triangularly-shaped obstacles disposed in amicrofluidic channel. In the figure, fluid flows in the left-to-rightdirection, as indicated by the arrow marked, “Fluid.” In this array,equilateral triangular posts are disposed in a parallelogram latticearrangement that is tilted with respect to the directions of fluid flow.Other lattice arrangements (e.g., square, rectangular, trapezoidal,hexagonal, etc. lattices) can also be used. The tilt angle ε (epsilon)is chosen so the device is periodic. In this embodiment, a tilt angle of18.4 degrees (⅓ radian) makes the device periodic after three rows. Thetilt angle ε also represents the angle by which the array direction isoffset from the fluid flow direction. The gap between posts is denoted Gwith equilateral triangle side length S. Streamlines are shown extendingbetween the posts, dividing the fluid flow between the posts into threeregions (“stream tubes”) of equal volumetric flow. A relatively largeparticle (having a size greater than the critical size for the array)follows the array tilt angle when fluid flow is in the direction shown.A relatively small particle (having a size smaller than the criticalsize for the array) follows the direction of fluid flow.

FIG. 6B is a comparison of normalized velocity flow between twoequilateral triangular posts (left panel) and normalized velocity flowbetween two circular posts (right panel). The shaded portions representan equal proportion of area-under-the-curve, demonstrating that thecritical radius for particles flowing past the point of the triangle issignificantly smaller (<15% gap width) than the critical radius forparticles flowing past the round post (>20% gap width).

FIG. 7 is a graph illustrating hypothetical and experimental effects ofthe tilt angle (“Array Tilt” in FIG. 7) on particle displacement.

FIG. 8 is a graph illustrating the effect of the tilt angle (“ArrayTilt” in FIG. 8) on gap length G. G_(T) refers to the gap length betweentriangular posts, and G_(C) refers to the gap length between roundposts.

FIG. 9 is a graph illustrating the effect of applied pressure onparticle velocity in bump arrays having triangular posts (data shown astriangles) and bump arrays having circular posts (data shown ascircles).

FIG. 10 is a graph illustrating the effect of obstacle edge roundness(expressed as r/S) on the critical size exhibited on the side of a gapbounded by the edge.

FIG. 11 is an image of an array constructed as described herein.

FIG. 12 illustrates particle motion in a ratchet bump array of the typedescribed herein.

FIG. 13 illustrates particle motion in a ratchet bump array of the typedescribed herein.

FIG. 14 illustrates particle motion in a ratchet bump array of the typedescribed herein.

FIG. 15 is a graph comparing the critical size characteristics of roundand triangular posts.

FIG. 16 shows conventional methods (left) for processing cells (e.g.,leukocytes) and two embodiments of the methods described herein(vertically down the center and vertically down the right)

FIG. 17A shows a DLD array designed to “bump” E. coli (>1 μm size).

FIG. 17B shows a DLD array having a first stream comprising a samplestream, and a second stream comprising a buffer.

FIG. 17C shows a time lapse image of leukocytes being concentrated andharvested from a DLD array similar to the DLD array shown in FIG. 17B.

FIG. 18A shows a multi-stream “car wash” chip for multiple sequentialchemical processing;

FIG. 18B shows false color fluorescent time lapse image of plateletsmoving downward in a DLD array.

FIG. 19 shows input and output views of a multi-steam “car wash” DLDarray for multiple sequential chemical and/or enzymatic processing.

FIG. 20A shows 10 μm beads flowing from an input, middle, and outputportion of a multi-stream “car wash” DLD array as shown in FIG. 19. FIG.20B shows 2 μm beads flowing from a middle, and output portion of amulti-stream “car wash” DLD array as shown in FIG. 19.

FIG. 21 shows a schematic of the diffusion between a reagent stream andan adjacent parallel flow stream as the parallel flow streams flowwithin a microfluidic channel.

FIG. 22 shows a schematic of a model of incubation time and limitedsource diffusion.

FIG. 23 shows the relationship between incubation time and flow speed(top) as well as the relationship between concentration and flow speed(bottom) in a “car wash” chip as depicted in FIGS. 18A and 19.

FIG. 24A shows a model of “car wash” chip comprising a sample stream,reagent stream, and buffer stream further comprising a pair of opposingseparator walls that extend into the interior of the bump array andseparate the reagent stream from the buffer stream. FIG. 24B showsinput, middle, and output views of a multi-steam “car wash” DLD array asdescribed in FIG. 24A comprising a pair of opposing separator walls thatextend into the array of obstacles for multiple sequential chemicalprocessing.

FIG. 25 shows the results of running a sample comprising 2 μm and 10 μmlabeled beads through a “car wash” DLD array comprising a pair ofopposing separator walls as depicted in FIGS. 24A and B.

FIG. 26 shows a comparison of contamination in the output portion of a“car wash” DLD array with (Modified) and without (Original) opposingseparator walls.

FIG. 27A shows a model of “car wash” chip comprising a sample stream,and 2 adjacent buffer streams further comprising a pair of opposingseparator walls that extend into the interior of the bump array andseparate the two buffer streams. FIG. 27B shows input, middle, andoutput views of a multi-steam “car wash” DLD array as described in FIG.27A comprising a pair of opposing separator walls that extend into thearray of obstacles for multiple sequential chemical processing.

FIG. 28 shows the results of running a blood sample through a “car wash”DLD array comprising a pair of opposing separator walls as depicted inFIGS. 27A and B.

FIG. 29A shows views of areas of potential clogging in the input, middleand output portions of the array of obstacles in the DLD array of FIG.28 following flowing a blood sample through the DLD array shown in FIG.28. FIG. 29B shows magnified views of areas of potential clogging in themiddle and output portions of the array of obstacles shown in FIG. 29A.

FIG. 30 shows the inputs and outputs of running a blood sample throughany of the “car wash” DLD arrays as provided herein.

FIG. 31 shows the relationship between incubation time and flow speed(top) as well as the relationship between concentration and flow speed(bottom) in a “car wash” chip with (Modified design; FIGS. 27A and B)and without (Ver. 1 chip; FIG. 19) a pair of opposing separator walls.

FIG. 32 shows a model of a “car wash” chip comprising a sample stream,two reagent streams, and two buffer streams further comprising threepairs of opposing separator walls that extend into the interior of thebump array and separate the reagent streams from the buffer streams.

FIG. 33A shows a DLD array with an on-chip cleaning system, whichcomprises inlets and outlets in the pair of opposing walls for fluidconfinement. FIG. 33B shows blocking of the on-chip cleaning systemwhile the DLD array is in the bump mode (e.g. sample or fluid comprisingparticles (e.g., cells) are flowing from left to right through DLDarray). FIG. 33C shows blocking of the channel comprising the DLD arrayduring cleaning of the DLD array using an on-chip cleaning system asdescribed herein.

FIG. 34A shows clogged beads in a DLD array comprising an on-chipcleaning system as depicted in FIGS. 33A-C following flowing of labeledbeads through the DLD array during the bump mode. FIG. 34B shows aportion of the DLD array following the bump mode before the cleaningmode (top) and after the cleaning mode (bottom).

FIG. 35 shows a simplified diagram of process by which platelet-inducedclogging of a DLD array as provided herein can occur.

FIG. 36 shows the results of experiments identifying calcium-dependentintegrins and thrombin-induced platelet activation as the dominantcontributors to platelet-induced clogging of DLD arrays as providedherein. The x-axis shows the volume of blood that has been processedthrough the array, while the y-axis shows the fluorescence of leukocytesstuck or clogged in the array. Diluted blood was actually processed, butthis x-axis represents the amount of undiluted blood that was usedbefore dilution and which flowed through the array, which had 40 microntriangular posts and a gap width of 27 microns.

FIG. 37 shows images of clogging in arrays with three differentparameters for (a) 1 mM EDTA and (b) 5 mM EDTA and 40 μM PPACK. Thevolume of blood through each channel and the flow rate was the same inboth (a) and (b). The flow direction was left to right. Green indicatedstuck or clogged leukocytes.

FIG. 38 shows the effect of flow rate and blood dilution on clogging ofa DLD array as provided herein.

FIG. 39 illustrates an embodiment of a device comprising an array ofobstacles comprising 14 channels.

FIG. 40 illustrates an embodiment of a device comprising an array ofobstacles comprising 2 channels.

FIG. 41 illustrates a desktop instrument and a disposable cellseparation module. Up to 8 cell separation modules can be used in thedesktop instrument, with up to 10 samples per module. The instrument canbe stand alone or can be integrated in-line with other equipment. Adisposable cell separation module can comprise a blood sample input port(in some cases with a safe enclosure feature), a micropost arraychamber, a product outlet port, a waste outlet port (in some cases withsafe on-board containment feature, and a buffer input port.

DETAILED DESCRIPTION I. Overview

Provided herein are methods, compositions, devices, systems, and kitsfor chemical processing, purification, isolation, and/or concentrationof particles. In some cases, the chemical processing, purification,isolation, and/or concentration of particles can be high-throughput. Thechemical processing, purification, isolation, and/or concentration ofparticles can involve separating particles based on size, e.g., flowinga sample through an array of obstacles, e.g., deterministic lateraldisplacement (DLD) array. Devices for separating particles based on sizeand/or using DLD are described, e.g., in U.S. Pat. Nos. 7,150,812,7,318,902, 7,472,794, 7,735,652, 7,988,840, 8,021,614, 8,282,799,8,304,230, 8,579,117, and PCT Publication No. WO2012094642, which areherein incorporated by reference in their entireties. In some cases, thehigh-throughput methods comprise flow rates of at least 1 mL/min, atleast 5 mL/min, at least 10 mL/min or at least 20 mL/min. In some cases,devices described herein can process less than 1 ml, at least 10 mL, atleast 100 mL, or at least 300 mL of sample.

In one aspect, a device for processing, purifying, and concentratingparticles is provided herein, wherein the device comprises a channelextending from a plurality of inlets to a plurality of outlets, andwherein the channel is bounded by a first wall and a second wallopposite from the first wall; and an array of obstacles disposed withinthe channel configured to deflect particles toward the second wall whenthe particles are flowed from the inlets to the outlets. The device canbe configured such that particles in a fluid sample are inputted into atleast one of the plurality of inlets and particles of a predeterminedsize in the fluid sample are deflected through a series of parallel flowstreams flowing from the plurality of inlets to the plurality of outletswhile also being deflected toward the second wall. In some cases, theseries of parallel flow streams comprises at least four flow streamscomprising a reagent. The reagents can be the same or differentreagents.

In another aspect, a device is provided herein, the device comprising achannel extending from a plurality of inlets to a plurality of outlets,wherein the channel is bounded by a first wall and a second wallopposite from the first wall, and wherein the device is configured toflow a plurality of flow streams from the plurality of inlets to theplurality of outlets, wherein the plurality of flow streams flowparallel to each other; and an array of obstacles disposed within thechannel configured to deflect particles toward the second wall when theparticles are flowed from the inlets to the outlets, wherein the arrayof obstacles comprises at least one separator wall oriented parallel toflow of the plurality of flow streams and the first and second walls.The device can be configured wherein particles introduced into an sampleinlet near the first wall pass through the plurality of flow streamswhile being deflected toward the second wall, and wherein the at leastone separator wall is configured to delay the flow of deflectedparticles toward the second wall, wherein the delay serves tosubstantially increase an amount of time that deflected particles residein a flow stream, and/or substantially reduce mixing between parallelflow streams.

In another aspect, a device is provided herein, the device comprising achannel extending from at least one inlet to a plurality of outlets,wherein the channel is bounded by a first wall and a second wallopposite from the first wall; and an array of obstacles disposed withinthe channel configured to deflect particles in a stream comprisingparticles toward the second wall when the stream comprising particles isflowed from the at least one inlet to the plurality of outlets. Thedevice is configured wherein the first wall comprises a plurality ofinlets adapted to flow a fluid towards a plurality of outlets in thesecond wall, wherein the direction of the flow of the fluid isperpendicular to flow of the stream comprising the particles and whereinthe flow of the fluid is configured to remove any particles that havebecome clogged in the array of obstacles following movement of theparticles toward the second wall.

In another aspect, a method for labeling cells is provided, the methodcomprising providing a sample comprising cells processing the samplecomprising cells, wherein the processing comprises introducing thesample comprising cells into a sample inlet of a device comprising anarray of obstacles, and passing the sample through the array ofobstacles, wherein the array of obstacles comprises a plurality ofparallel flow streams flowing through the array of obstacles, whereinthe passing comprises flowing cells from the sample from the sampleinlet through the plurality of parallel flow streams, wherein at leastone of the plurality of parallel flow streams comprises a labelingreagent, at least one of the plurality of parallel flow streamscomprises a fixation agent, at least one of the plurality of parallelflow streams comprises a permeabilization agent, and at least one of theplurality of parallel flow streams comprises a wash buffer, whereby thepassing the sample through the array of obstacles serves to label thecells while also simultaneously separating the cells by size; andharvesting labeled cells of a predetermined size from one of a pluralityof outlets of the device.

In another aspect, a method for processing leukocytes for moleculardiagnostic testing is provided, the method comprising labeling andharvesting the leukocytes from a sample using a microfluidic device,wherein the yield of labeled cells is at least 85% and the viability ofthe labeled cells is at least 90%.

In another aspect, a system for processing and analyzing particles isprovided, the system comprising a plurality of reservoirs, wherein atleast one of the reservoirs comprises a sample comprising particles, andat least one of the reservoirs comprises a reagent; a device, whereinthe device is in fluid communication with each of the plurality ofreservoirs, and wherein the device is adapted to process particles fromthe sample comprising particles, wherein the processing comprisesflowing the sample comprising particles from the reservoir comprisingthe sample into an input of a device, and passing the particles throughthe device, wherein the passing comprises flowing the particles from theinput through a plurality of parallel flow streams within the device,wherein at least one of the parallel flow streams comprises a reagentwhich flows from at least one of the plurality of reservoirs, andwherein the device comprises an array of obstacles, whereby the passingthe particles through the device serves to process the particles as wellas separate the particles by size; and an analytical device in fluidcommunication with at least one of a plurality of outlet ports of thedevice, wherein the analytical device is configured to perform ananalysis of particles processed by the device.

Microfluidic processes known as Deterministic Lateral Displacement (DLD)can remove cells from a flow of fluid, on the basis of their size (2).As a mixture of fluid and particles flows through an array ofmicroposts, in which the micropost axis can be tilted at a small angleof a few degrees from the direction of the fluid flow, particles above acertain critical size (such as leukocytes) can “bump” off the posts toflow in a direction along the tilted array axis (hence the device can bereferred to as a “bump array”). Smaller particles and dissolvedmolecules, such as red blood cells, monoclonal antibodies (Mabs), andchemical reagents (e.g., fixatives, enzymes, permeabilization agents)can flow straight ahead, on average, with or in the fluid stream. Thus,after travelling across the microfluidic chip, the larger cells can beflowed out of and away from the fluid stream of the original inputmixture and can be collected separately. The process can be used toremove a range of objects from an input fluid, ranging from large DNAoligomers (˜100 kpb) to E. coli and other bacteria, platelets,erythrocytes and leukocytes (2,4,5). The critical size determining whichpath the cells or other objects follow can be controlled by the designof the micropost array (e.g. post size and shape, gaps between posts,axis tilt angle) (6). Cells or particles several times larger than thecritical size that determines bumping (i.e. cell harvest) can flowthrough the device without clogging. In some cases, the operatingconditions (e.g. chip loading, flow rates, output collection) can beautomated.

No previously existing cell processing method can recover all subsets ofleukocytes in >90% yield, which can be a performance criteria that canbe achieved using a microfluidic device and methods as provided herein.This methods and devices can be a research, clinical, and/or commercialinnovation that can replace the current standard centrifugalwash/concentrate steps that can be commonplace in research and clinicallaboratories. The use of this cell processing procedure may not berestricted to flow cytometry and/or to leukocytes.

The tests to quantify the numbers and functional states of key leukocytetypes from blood samples can offer enhanced precision andpersonalization of clinical diagnosis, prognosis, and treatmentresponse. For example, labeling of >30 cell surface and intracellulartarget molecules can assess signaling pathway status of multiple typesof normal leukocytes vs. leukemia cells simultaneously, bymulti-parameter flow cytometry or atomic mass spectrometry (1). Stemcells or infected cells can be analyzed similarly. However, currentprocedures to process blood leukocytes can be expensive, time-consuming,repetitive, and human operator-dependent; and can have low cell yieldsand can require considerable human expertise.

Conventionally, combined surface membrane and intracellular labeling ofblood leukocytes can require lysing erythrocytes to harvest theleukocyte population (Lysis); incubating with fluorescent monoclonalantibodies (Mabs) against cell surface leukocyte lineage/stage or cancermarkers (Surface Labeling); performing a fixation/permeabilization(Fix/Perm) step; and incubating with reagents (e.g. tagged Mabs, nucleicacids, dyes) that can bind to intracellular (cytoplasmic and nuclear)molecules (Intracellular Labeling). Following each of these 4 steps, oneor more Wash/Concentrate steps can be required, currently involvingcentrifugation and resuspension of the cell pellet. Leukocyte yield canbe ˜80-90% in each Wash/Concentrate step, so overall yield can be <50%after multiple washes.

Described herein are methods, compositions, devices, system and/or kitsutilizing Deterministic Lateral Displacement (DLD) microfluidictechnology (2) to replace each of the Wash/Concentrate Steps. In somecases, a leukocyte harvesting and a Wash/Concentrate step is combinedinto a single step, thereby avoiding lysis and further streamliningworkflow. Thus, the current multi-step, labor-intensive process takingup to a half-day can be replaced by a high yield, low cost process thattakes <1 hr and has less or minimal operator-dependent steps. In somecases, the multiple sequential steps can be performed to harvest, label,and wash/concentrate leukocytes in a “Car Wash” approach on a singlemicrochip, inputting whole blood and outputting labeled cells fordownstream applications (e.g., flow cytometry or mass spectrometryanalysis).

The Deterministic Lateral Displacement (DLD) separation described herecan outperform standard centrifugal procedures. Provided herein aremicrofluidic devices, compositions, systems, kits, and methods to washand concentrate leukocytes rapidly, at low cost, with increased cellyield, and with improved reproducibility. Microfluidic DLD systems canbe designed and fabricated to remove and concentrate leukocytes orspiked leukemia cells from a stream containing Mabs used for theLabeling steps or from the solution used for a Fix/Perm step. Volumes of0.1-1 ml can be processed in <5-10 minutes (e.g., by an automatedprocess). In some cases, >90% yield and 90% viability of leukocytes andremoval of >99% unbound fluorescent or Fix/Perm reagents with no skewingof sub-populations is achieved.

In some cases, an erythrocyte Lysis step and subsequent centrifugalWash/Concentrate step is replaced by a DLD microfluidic step. In somecases, the DLD microfluidic step is a single DLD microfluidic step.

As described herein, leukocytes can be labeled in whole blood (healthyand leukemia samples), and then a DLD microfluidic process can isolate,purify, and/or concentrate the Mab-labeled leukocytes from the mixtureof blood and excess free Mab. In some cases, >99% erythrocyte depletionis achieved.

The devices, methods, compositions, and/or kits can prepare leukocytes(including leukemia cells in blood) for flow cytometry. In some cases,the devices, methods, compositions, systems, and/or kits provided hereinare a general replacement for centrifugation in preparative proceduresfor diverse tests to be performed on samples. The diverse tests can beany test or downstream application as described herein. The samples canbe any sample as provided herein (e.g., on blood leukocytes).

II. Particles

In some cases, a particle that can be chemically and/or enyzmaticallyprocessed or treated, purified, isolated, and/or concentrated usingmethods, compositions, devices, systems, and/or kits described hereincan be a cell, cellular fragment, or nucleic acid. In some cases, aparticle is not a cell, cellular fragment, or nucleic acid, e.g., aparticle is a bead.

A. Blood Components

In some cases, a particle is a blood component. In some cases, methods,compositions, devices, systems, and/or kits described herein can be usedto process (e.g., chemically and/or enzymatically process or treat),purify or separate blood components, e.g., for blood banking. In somecases, a blood component comprises a platelet, red blood cell(erythrocyte), white blood cell (e.g, granulocytes, e.g, neutrophil,basophil, or eosinophil, or agranulocyte, e.g., lymphocyte, monocyte, ormacrophage). In some cases, methods described herein can be used forleukocyte depletion from red blood cells or platelets (e.g., to replaceleukocyte filters in processing of blood products for transfusion ofpatients). In some cases, methods described herein can be used forin-line leukocyte or platelet isolation (e.g., to replace centrifugalapheresis). In some cases, methods, compositions, devices, systems,and/or kits described herein can be used to deplete erythrocytes from ablood sample, e.g., an umbilical cord blood sample.

In some cases, a cell is a dendritic cell. In some cases, a cell is anycell of the innate or adaptive immune system.

B. Leukocytes (White Blood Cells)

In some cases, a cell processed (e.g., chemically and/or enzymaticallyprocessed or treated), purified, isolated, and/or concentrated usingmethods, compositions, devices, systems, and/or kits described herein isa leukocyte (white blood cell). A leukocyte can be, e.g, a neutrophil,eosinophil, basophil, lymphocyte, or monocyte. A leukocyte can be, e.g,a granulocyte or agranulocyte. In some cases, a granulocyte is aneutrophil, basophil, or eosinophil. In some cases, an agranulocyte is alymphocyte, monocyte, or macrophage. A lymphocyte can be, e.g., a B-cellor a T-cell. A T-cell can be, e.g., a CD4+ T helper cell (e.g, T_(H)1,T_(H)2, T_(H)3, T_(H)17, T_(H)9, or T_(FH)), a CD8+ cytotoxic T-cell, aγδ T cell, a regulatory (suppressor) T-cell, a Natural Killer T (NKT)cell, an or antigen-specific T-cell, e.g., memory T cell, e.g., centralmemory T-cells, T_(EM) cells, or T_(EMRA) cell. In some cases, alymphocyte is a Natural Killer (NK) cell. A B-cell can be a plasmaB-cell, a memory B-cell, a B-1 cell, a B-2 cell, a marginal-zone B-cell,a follicular B-cell, or a regulatory B-cell. In some cases, a cell is aregulatory macrophage. In some cases, a cell is a plasmacytoid dendriticcell (pDC). In some cases, a cell is myeloid-derived suppressor cells(MDSCs). In some cases, a cell is a megakarocyte.

In some cases, a leukocyte (white blood cell) can be identified by oneor more cell surface markers. In some cases, a leukocyte (white bloodcell) can be identified through the use of binding agents (e.g.,antibodies, antibody fragments, probes, etc. as provided herein) to oneor more cell surface markers (e.g., detecting the presence and/orabsence of a label associated with the binding agent). The cell surfacemarker on a leukocyte (human or mouse) can be a transmembrane protein.The transmembrane protein can be a glycoprotein. In some cases, atransmembrane glycoprotein used to identify a leukocyte (e.g., human ormouse) is a cluster of differentiation (CD) transmembrane glycoprotein.The binding agent can comprise a label as provided herein. The label asprovided herein can be detectable (e.g., fluorescence, etc.) The CDprotein can be CD1a, 1b, 1c, 1d, 2, 3, 4, 5, 8, 10, 11a, 11b, 11c, 13,14, 15, 16/32, 19, 20, 21/35 (CR2/CR1), 22, 23, 25, 26, 31, 33, 38, 39,40, 44, 45RB, 45RA, 45R/B220, 49b (pan-NK cells), 49d, 52, 53, 54, 57,62L, 63, 64, 66b, 68, 69, 70, 73, 79a (Igα), 79b (Igβ), 80, 83,85g/ILT7, 86, 88, 93, 94, 103, 105 (Endoglin), 107a, 107 (Mac3), 114,115, 117, 119, 122, 123, 124, 127, 129, 134, 137(4-1BB), 138(Syndecan-1), 158 (Kir), 161, 163, 183, 184 (CXCR4), 191, 193 (CCR3),194 (CCR4), 195, 195 (CCR5), 197, 197 (CCR7), w198 (CCR8), 203c,205/Dec-205, 207 (Langerin), 209DC-SIGN), 223, 244 (2B4), 252 (OX40L),267, 268 (BAFF-R), 273 (B7-DC, PD-L2), 278 (ICOS), 279/PD-1, 282 (TLR2),289 (TLR9), 284 (TLR4), 294, 303, 304, 305, 314 (NKG2D), 319 (CRACC),328 (Siglec-7), or 335 (NKp46). A leukocyte (white blood cell) cellsurface marker can be, e.g., surface IgM, IgD, DC Marker (33D1), F4/80,CMKLR-1, HLA-DR, Siglex H, MHC Class II, LAP (TGF-b), GITR, GARP, FR4,CTLA-4, TRANCE, TNF-β, TNF-α, Tim-3, LT-βR, IL-18R, CCR1, TGF-β, IL-1R,CCR6, CCR4, CRTH2, IFN-γR, Tim-1, Vα24-Jα18 TCR (iNKT), Ly108, Ly49,CD56 (NCAM), TCR-α/β, TCR-γ/δ, CXCR1, CXCR2, GR-1, JAML, TLR2, CCR2,Ly-6C, Ly-6G, F4/80, VEGFR1, C3AR, FcεRIα, Galectin-9, MRP-14, Siglec-8,Siglec-10. TLR4, IgE, GITRL, HLA-DR, ILT-3, Mac-2 (Galectin-3). CMKLR-1,or DC Marker (33D1).

In some cases, a leukocyte (white blood cell) can be identified by oneor more intracellular markers. A leukocyte (white blood cell)intracellular marker can be, e.g., Pax-5, Helios, FoxP3, GM-CSF, IL-2,IFN-γ, T-bet, IL-21, IL-17A, IL-17F, IL-22, RORγt, RORα, STAT3, IL-10,IL-13, IL-5, IL-4, IL-6, GATA3, c-Maf, Granzyme B, Perforin, orGranulysin.

In some cases, the devices, method, compositions, systems, and/or kitsprovided herein can be used to differentiate between mature and immatureB and T cells. As provided herein, differentiation or identification ofthe presence (e.g., positive), absence (e.g., negative) and/or acombination thereof a marker as provided herein can be determinedthrough the use of a binding agent specific for or directed to oragainst the marker (e.g., cell surface and/or intracellular). Thebinding agent can be any binding agent known in the art (e.g., antibody,antibody fragment, probe (e.g., nucleic acid probe), etc.). The bindingagent can comprise a label as provided herein. The label as providedherein can be detectable (e.g., fluorescence, etc.). Differentiationbetween mature and immature T cells can be performed by identifying thepresence (e.g., positive), absence (e.g., negative) and/or a combinationthereof of CD3, 4, 8, 25, 44, 117, 127, and TCR-α/β, TCR-γ/δ markers(e.g., detecting the presence and/or absence of a label associated witha binding agent). Differentiation between mature and immature Treg cellscan be performed by identifying the presence (e.g., positive), absence(e.g., negative) and/or a combination thereof of CD3, 4, 8, 25, 40, 44,45RA, 45RB, 62L, 117, 127, 134 (OX40), 137 (4-1BB), 197, 199 (CCR9), 223(LAG-3), Integrin α4, Integrin β7, 304 (Neuropilin), 357(GITR), CTLA-4,Foxp3, GARP, IL-10, IL-15Rα, LAP (TGFβ) and TCR-α/β markers.Differentiation between mature and immature mouse B cells can beperformed by identifying the presence (e.g., positive), absence (e.g.,negative) and/or a combination thereof of CD2, 11b, 16/32, 19, 21/35,23, 22, 24 (HAS), 43, 45R (B220), 93 (AA4.1), 117, 127, BP-1, cμ, sμ,

5, IgD, IgM, markers. Differentiation between mature and immature humanB cells can be performed by identifying the presence (e.g., positive),absence (e.g., negative) and/or a combination thereof of CD9, 19, 20,23, 24 (HSA), 27, 28, 31, 34, 38, 40, 45R (B220), 95 (FAS), 150 (SLAM),184 (CXCR4), IgD, IgM, IgA, IgG markers. Differentiation between matureand immature human or mouse dendritic cells can be performed byidentifying the presence (e.g., positive), absence (e.g., negative)and/or a combination thereof of CD3, 11b, 11c, 14, 16, 19, 34, 49b, 68,80, 86, 107b (Mac-3), 115, 117, 123, 127, 135, 303 (BDCA-2), CXCR1,F4/80, Ly6C, Ly6G, Gr-1, MHC II, NKp46, Sca-1, Ter119, iNOS, TNFα,markers. Differentiation between mature and immature human or mousemonocytes/macrophages can be performed by identifying the presence(e.g., positive), absence (e.g., negative) and/or a combination thereofof CD3, 11b, 11c, 13, 14, 16, 19, 33, 34, 43, 45, 62L, 68, 115, 117,123, 127, 135, 163, 204, 206, CCR2, CCR5, CX3CR1, F4/80, LysA/E, Tie-2,Lys6B.2, Ly6C, Ly6G, Gr-1, MHC II, NKp46, Sca-1, Ter119, VEGFR1 markers.Differentiation between mature and immature human and mouse macrophagesubclasses can be performed by the presence (e.g., positive), absence(e.g., negative) and/or a combination thereof of a number of proteinsincluding CD14, CD11b, 16, 32, 51/61, 64, 68, 86, 121a, 121b, 124, 150,163, 181, 182, 206, 210, 215, CCLs 1, 2, 3, 4, 5, 8, 15, 16, 18, 20, 24,CCR2, CCR7, CXCLs, 8, 9, 10, 11, 13, 16, IFNγ, IL-1RA, IL-1β, IL-6,IL-10, IL-12, IL-15, IL-23, iNOS, Ly6C, MHC II (human and mouse), TNF-α,Dectin-1, FcεRlα, Lectins, RAGE, Scavenger Receptor, Arginase, or LAP(TGFβ) markers.

In some cases, the devices, method, compositions, systems, and/or kitsprovided herein can be used to differentiate between or identify subsetsof leukocytes (white blood cells). Differentiation or identification ofsubsets of leukocytes (white blood cells) can be through treating asample comprising particles (e.g., leukocytes) with one or more bindingagents as described herein specific for or directed to or against one ormore of the markers (e.g., cell surface or intracellular markers). Thebinding agent can comprise a label as provided herein. The label asprovided herein can be detectable (e.g., fluorescence, etc.) Subsets ofleukocytes (white blood cells) can be differentiated from otherleukocytes through identifying the presence (e.g., positive), absence(e.g., negative), and/or combinations of leukocyte-type specificintracellular markers (e.g., detecting the presence and/or absence of alabel associated with a binding agent). Subsets of leukocytes (whiteblood cells) can be differentiated from other leukocytes throughidentifying the presence (e.g., positive), absence (e.g., negative),and/or combinations of leukocyte-type specific markers. For example, Th2cells can be identified by the transcription factor GATA3 and thecytokines: IL-4, IL-5, IL-6, IL-10, and IL-13. Th17 cells can beidentified by intracellular RORγt and STAT3 and production of cytokines:IL-17A, IL-17F, IL-21 and IL-22. Th1 cells can be identified by thetranscription factor T-bet and cytokines: IL-2, IFN-γ, and TNF. Amegakaryocyte can be identified by GM-CSF, IL-3, IL-6, IL-11, chemokines(SDF-1; FGF-4), and erythropoietin. NK can be identified by the presence(e.g., positive), absence (e.g., negative) and/or a combination thereofof cell surface markers CD16 (FcγRIII) and CD56 in humans, NK1.1 orNK1.2 in C57BL/6 mice, while they do not express T-cell antigenreceptors (TCR) or Pan T marker CD3 or immunoglobulins (Ig) B cellreceptors, which are found on T and B cells. Up to 80% of human NK cellsalso express CD8. They are distinct from Natural Killer T cells, whichexpress CD3. In humans, pDCs express the surface markers CD123,BDCA-2(CD303) and BDCA-4(CD304), but do not express CD11c or CD14, whichdistinguishes them from conventional dendritic cells or monocytes,respectively. Mouse pDC express CD11c, B220, BST-2 (mPDCA) and Siglec-Hand are negative for CD11b. T regulatory cells can be characterized oridentified by the presence (e.g., positive), absence (e.g., negative)and/or a combination thereof of CD4, CD25, and Foxp3, while lackingCD127. CD4+Foxp3+ regulatory T cells have been referred to as“naturally-occurring” regulatory T cells to distinguish them from“suppressor” T cell populations that are generated in vitro. While othervariants of suppressive T cells do exist, such as CD8 suppressor cells,Th3 and Tr-1 cells, Tregs are classically defined as CD4+CD25+FOXP3+cells. Macrophages can be identified by specific expression of a numberof proteins including CD14, CD11b, F4/80 (mice)/EMR1 (human),MAC-1/MAC-3 and CD68. Myeloid-derived suppressor cells (MDSCs) are aheterogeneous population of cells that have potent T cell-suppressivefunction, characterized by an activated state with increased productionof reactive oxygen and nitrogen species, and arginase 1. In mice, MDSCscan be identified by CD11b+GR1+, while in humans, MDSCs can beidentified by LIN−HLA−DR−CD33+ or CD11b+CD14−CD33+. Th17 cells can beidentified using cell surface markers for CD4, CD161 and CCR6.

C. Stem Cells

In some cases, a cell processed (e.g., chemically or enzymaticallyprocessed or treated), purified, isolated, and/or concentrated usingmethods, compositions, devices, systems, and/or kits described herein isa stem cell. In some cases, the stem cell is an adult stem cell (somaticstem cell). In some cases, an adult stem cell is a hematopoietic stemcell (HSC) or hematopoietic progenitor cell (HPC). In some cases, a HSCis from bone marrow (e.g., bone marrow of pelvis, femur, or sternum). Insome cases, an HSC is in a cord blood sample, e.g., umbilical cordblood. In some cases, an HSC is from placenta. In some cases,granulocyte-colony stimulating factor (G-CSF) is administered to asubject; G-CSF can induce HSCs to leave bone marrow and circulate inblood vessels. In some cases, an HSC is in peripheral blood (e.g., G-CSFmobilized adult peripheral blood). In some cases, a stem cell is along-term stem cell or a short-term progenitor cell. In some cases, stemcells are used for ex vivo expansion, and the products of ex vivoexpansion are purified using methods and devices described herein. Insome cases, a stem cell is derived from adipose tissue (adipose-derivedstem cells (ASCs)). In some cases, stem cells are derived from acollengase digest of adipose tissue.

In some cases, a HSC (e.g., undifferentiated HSC) can be identified byone or more cell surface markers. In some cases, characterizing oridentifying a HSC (e.g., undifferentiated HSC) comprises identifying thepresence (e.g., positive), absence (e.g., negative), and/or combinationsof one or more cell surface markers. The identifying can be throughtreating a sample comprising particles (e.g., HSCs) with one or morebinding agents as described herein specific for or directed to oragainst one or more of the markers (e.g., cell surface). The bindingagent can comprise a label as provided herein. The label as providedherein can be detectable (e.g., fluorescence, etc.). The identificationcan be through identifying the presence (e.g., positive), absence (e.g.,negative), and/or combinations of the markers (e.g., detecting thepresence and/or absence of a label associated with a binding agent). Themarker can be a cell surface or an intracellular marker. The bindingagent can be any binding agent known in the art (e.g., antibody,antibody fragment, probe (e.g., nucleic acid probe), etc.). A cellsurface marker on a HSC can be a cluster of differentiation (CD)transmembrane glycoprotein. The CD can be CD 10, 31, 34, 38, 44, 45, 59,84, 90, 93 (C1Rqp), 105 (Endoglin), 110, 111, 117 (c-Kit), 133, 135(Flk-2), 150 (SLAM), 184 (CXCR4), 202b (Tie2/Tek), 243 9MDR-1), 271(NGFR), 309 (VEGFR2), 338. A cell surface marker on a HSC can be aHematopoietic Stem Cell Marker, VEGF Receptor 2, CXCR4, AngiotensinConverting Enzyme 1, BCRP/ABCG2, Ly-6A/E (Sca-1). A human HSC cellsurface marker can be, e.g., CD34+, CD59+*, Thy1⁺, CD38^(low/−),C-kit^(−/low), or lin⁻. A mouse HSC cell surface marker can be, e.g.,CD34low/−, SCA-1+, Thy1^(+/low), CD38+, C-kit+, or lin−. Anintracellular marker can be e.g., ATF2, GATA1, GATA2, GFI1, orRUNX1/AML1.

An HSC can give rise to blood cells, e.g., red blood cells, Blymphocytes, T lymphocytes, natural killer cells, neutrophils,basophils, eosinophils, monocytes, and macrophages.

An adult stem cell (somatic stem cell) can be an HSC, a mesenchymal stemcell, a neural stem cell, an epithelial stem cell, or a skin stem cell.In some cases, a stem cell is a mesenchymal stem cell. A mesenchymalstem cell can give rise to, e.g., bone cells (osteocytes), cartilagecells (chondrocytes), fat cells (adipocytes), and other kinds ofconnective tissue cells such as those in tendons.

In some cases, a stem cell is neural stem cell. A neural stem cell canbe found in a brain. A neural stem cell can give rise to, e.g., nervecells (neurons) and two categories of non-neuronal cells, e.g.,astrocytes and oligodendrocytes. In some cases, the devices, method,compositions, systems, and/or kits provided herein can be used toidentify a neural stem cell. Identification of a neural stem cell can bethrough treating a sample comprising particles (e.g., neural stem cell)with one or more binding agents as described herein specific for ordirected to or against one or more markers (e.g., cell surface orintracellular markers) expressed by neural stem cells. The binding agentcan comprise a label as provided herein. The label as provided hereincan be detectable (e.g., fluorescence, etc.). The identification can bethrough identifying the presence (e.g., positive), absence (e.g.,negative), and/or combinations of the markers (e.g., detecting thepresence and/or absence of a label associated with a binding agent). Themarkers can be cell surface markers, e.g., CD 15, 24, or 184. Themarkers can be intracellular markers, e.g., Nestin, Pax6, Sox1, or Sox2.

In some cases, a stem cell is an intestinal epithelial stem cell. Anintestinal epithelial stem cell can line the digestive tract and canoccur in deep crypts. An intestinal epithelial stem cell can give riseto absorptive cells, goblet cells, paneth cells, and/or enteroendocrinecells. In some cases, the devices, method, compositions, systems, and/orkits provided herein can be used to identify an intestinal epithelialstem cell. Identification of an intestinal epithelial cell can bethrough treating a sample comprising particles (e.g., an intestinalepithelial stem cell) with one or more binding agents as describedherein specific for or directed to or against one or more markers (e.g.,cell surface or intracellular markers) expressed by an intestinalepithelial stem cells. The binding agent can comprise a label asprovided herein. The label as provided herein can be detectable (e.g.,fluorescence, etc.). The identification can be through identifying thepresence (e.g., positive), absence (e.g., negative), and/or combinationsof the markers (e.g., detecting the presence and/or absence of a labelassociated with a binding agent). The markers can be cell surface orintracellular markers known in the art. The markers can be, e.g., CD 44,ICAM-1/CD54, CD34, Lrig1, Lgr5, Bmi1, Tert, or Hopx.

In some cases, a stem cell is skin stem cell. A skin stem cell can occurin the basal layer of epidermis and at the base of hair follicles. Anepidermal stem cell can give rise to keratinocytes, which can migrate tothe surface of the skin and form a protective layer. Follicular stemcells can give rise to both the hair follicle and to the epidermis. Insome cases, the devices, method, compositions, systems, and/or kitsprovided herein can be used to identify a skin stem cell. Identificationof a skin stem cell can be through treating a sample comprisingparticles (e.g., skin stem cell) with one or more binding agents asdescribed herein specific for or directed to or against one or moremarkers (e.g., cell surface or intracellular markers) expressed by skinstem cells. The binding agent can comprise a label as provided herein.The label as provided herein can be detectable (e.g., fluorescence,etc.). The identification can be through identifying the presence (e.g.,positive), absence (e.g., negative), and/or combinations of the markers(e.g., detecting the presence and/or absence of a label associated witha binding agent). The markers can be cell surface or intracellularmarkers known in the art. The markers can be, e.g., CD 34, K15, Nestin,Follistatin, p63, Integrin alpha 6, Tenascin C, EGFR, IGFR, Delta 1,TBRII, or Frizzled factors.

In some cases, a stem cell is an embryonic stem (ES) cell. An embryonicstem cell can be derived from embryos that develop from an egg that hasbeen fertilized in vitro. In some cases, an embryonic stem is a humanembryonic stem cell. In some cases, a stem cell is an inducedpluripotent stem cell (iPSC). An iPSC can be a somatic cell that isgenetically reprogrammed to an embryonic stem cell-like state. In somecases, a stem cell is an undifferentiated stem cell. In some cases, astem cell is cancer stem cell. In some cases, the devices, method,compositions, systems, and/or kits provided herein can be used toidentify an ES cell. Identification of an ES cell can be throughtreating a sample comprising particles (e.g., ES cell) with one or morebinding agents as described herein specific for or directed to oragainst one or more markers (e.g., cell surface or intracellularmarkers) expressed by ES cells. The binding agent can comprise a labelas provided herein. The label as provided herein can be detectable(e.g., fluorescence, etc.). The identification can be throughidentifying the presence (e.g., positive), absence (e.g., negative),and/or combinations of the markers (e.g., detecting the presence and/orabsence of a label associated with a binding agent). The markers can becell surface markers, e.g., CD 9, 15 (SSEA-1), 49f, 24, 29, 324, 338,SSEA-3, SSEA-4, SSEA-5, TRA-1-81, TRA-1-60, TRA-2-49, or TRA-2-54. Themarkers can be an intracellular marker, e.g., c-Myc, Nanog, FOXD3,OCT3/4. Stat-3, or Sox2.

In some cases, the devices, method, compositions, systems, and/or kitsprovided herein can be used to identify a mesenchymal stem cell.Identification of a mesenchymal stem cell can be through treating asample comprising particles (e.g., mesenchymal stem cell) with one ormore binding agents as described herein specific for or directed to oragainst one or more markers (e.g., cell surface or intracellularmarkers) expressed by mesenchymal stem cells. The binding agent cancomprise a label as provided herein. The label as provided herein can bedetectable (e.g., fluorescence, etc.). The identification can be throughidentifying the presence (e.g., positive), absence (e.g., negative),and/or combinations of the markers (e.g., detecting the presence and/orabsence of a label associated with a binding agent). The markers can becell surface markers, e.g., CD 10, 13, 29, 31, 44, 45RO, 49a, 49e, 51,54, 56, 73, 90, 105 (Endoglin), 106 (VCAM-1), 117 (c-kit), 166 (ALCAM),349 (Frizzled-9), TfR, BMPRs-IA, IB, and II, N-cadherin, SSEA-4, STRO-1,Sca-1/Ly6, PDGF R alpha, HLA class I, Carcino Embryonic Antigen,Integrin 5α, Integrin β1, p75, NGF Receptor, Sprouty 2, or TNAP. Themarkers can be intracellular markers, e.g., Bone alkaline phosphatase,dbx2, Flightless 1, KLF5, Spi1, Tafazzin, nucleotstemin, or vimentin.

D. Other Particles

In some cases, a particle that can be processed (e.g., chemically and/orenzymatically processed or treated), purified, isolated, and/orconcentrated using methods, compositions, devices, systems, and/or kitsdescribed herein can be a cancer cell, a circulating tumor cell (CTC),an epithelial cell, a circulating endothelial cell (CEC), a circulatingstem cell (CSC), or cancer stem cells. In some cases, a particle is abone marrow cell, progenitor cell foam cell, fetal cell, mesenchymalcell, circulating epithelial cell, circulating endometrial cell,trophoblast, immune system cell (host or graft), connective tissue cell,bacterium, fungus, virus, protozoan, algae, or plant cell. In somecases, a particle is a microparticle.

In some cases, a particle is a cellular fragment. In some cases, acellular fragment is a protein. In some cases, a protein is an antibody,or antibody fragment. In some cases, a cellular fragment is a T-cellreceptor. In some cases, a protein is an immunoglobulin. In some cases,a particle is a polypeptide.

In some cases, a particle is a rare cell, e.g., a cell type with anabundance of less than 1000 in a one mL sample, e.g., circulating tumorcells (CTCs), circulating fetal cells, stem cells, or cells infected bya virus or parasite. If sample is a water sample, a rare cell can be apathogenic bacterium or cell infected with a virus.

In some cases, a cellular fragment is a nucleic acid. A nucleic acid canbe, e.g., DNA or RNA. DNA can be genomic DNA, mitochondrial DNA, and/orcell-free DNA. RNA can be, e.g., messenger RNA (mRNA), ribosomal RNA(rRNA), transfer RNA (tRNA), signal recognition particle RNA, smallnuclear RNA, small nucleoar RNA, SmY RNA, small cajal body-specific RNA,telomerase RNA, spliced leader RNA, antisense RNA, CRISPR RNA, longnoncoding RNA (long ncRNA), microRNA (miRNA), short interfering RNA(siRNA), short hairpin RNA (shRNA), trans-acting siRNA, repeatassociated siRNA, and/or cell-free RNA. In some cases, a nucleic acid isdouble stranded. In some cases, a nucleic acid is single stranded. Insome cases, a nucleic acid comprises one or two overhangs. In somecases, a nucleic acid comprises a 5′ overhang. In some cases, a nucleicacid comprises a 3′ overhang. In some cases, the nucleic acid comprises“high molecular weight” nucleic acid. In some cases, a nucleic acid is alow molecular weight nucleic acid. In some cases, the nucleic acid isintranuclear, intracellular, or extracellular.

The term “polynucleotide”, “nucleic acid”, or grammatical equivalents,can refer to two or more nucleotides covalently linked together. Anucleic acid described herein can contain phosphodiester bonds, althoughin some cases, as outlined below (for example in the construction ofprimers and probes such as label probes), nucleic acid analogs areincluded that can have alternate backbones, comprising, for example,phosphoramide (Beaucage et al., Tetrahedron 49(10):1925 (1993) andreferences therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl etal., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res.14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al.,J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta26:141 91986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437(1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al.,J. Am. Chem. Soc. 111:2321 (1989), O-methylphosphoroamidite linkages(see Eckstein, Oligonucleotides and Analogues: A Practical Approach,Oxford University Press), and peptide nucleic acid (also referred toherein as “PNA”) backbones and linkages (see Egholm, J. Am. Chem. Soc.114:1895 (1992); Meier et al., Chem. Int. Ed. Engl. 31:1008 (1992);Nielsen, Nature, 365:566 (1993); Carlsson et al., Nature 380:207 (1996),all of which are incorporated by reference). Other analog nucleic acidsinclude those with bicyclic structures including locked nucleic acids(also referred to herein as “LNA”), Koshkin et al., J. Am. Chem. Soc.120.13252 3 (1998); positive backbones (Denpcy et al., Proc. Natl. Acad.Sci. USA 92:6097 (1995); non-ionic backbones (U.S. Pat. Nos. 5,386,023,5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al., Angew.Chem. Intl. Ed. English 30:423 (1991); Letsinger et al., J. Am. Chem.Soc. 110:4470 (1988); Letsinger et al., Nucleoside & Nucleotide 13:1597(1994); Chapters 2 and 3, ASC Symposium Series 580, “CarbohydrateModifications in Antisense Research”, Ed. Y. S. Sanghui and P. Dan Cook;Mesmaeker et al., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffset al., J. Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743(1996)) and non-ribose backbones, including those described in U.S. Pat.Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series580, “Carbohydrate Modifications in Antisense Research”, Ed. Y. S.Sanghui and P. Dan Cook. Nucleic acids containing one or morecarbocyclic sugars are also included within the definition of nucleicacids (see Jenkins et al., Chem. Soc. Rev. (1995) pp 169 176). Severalnucleic acid analogs are described in Rawls, C & E News Jun. 2, 1997page 35. “Locked nucleic acids” are also included within the definitionof nucleic acid analogs. LNAs can be a class of nucleic acid analoguesin which the ribose ring is “locked” by a methylene bridge connectingthe 2′-O atom with the 4′-C atom. All of these references are herebyexpressly incorporated by reference. These modifications of theribose-phosphate backbone can be done to increase the stability andhalf-life of such molecules in physiological environments. For example,PNA:DNA and LNA-DNA hybrids can exhibit higher stability and thus can beused in some embodiments. Nucleic acids can be single stranded or doublestranded, as specified, or contain portions of both double stranded orsingle stranded sequence. Depending on the application, the nucleicacids can be DNA (including, e.g., genomic DNA, mitochondrial DNA, andcDNA), RNA (including, e.g., mRNA and rRNA) or a hybrid, where thenucleic acid contains any combination of deoxyribo- andribo-nucleotides, and any combination of bases, including uracil,adenine, thymine, cytosine, guanine, inosine, xathanine hypoxathanine,isocytosine, isoguanine, etc.

In some cases, a cellular fragment is a membrane, cellular organelle,nucleosome, exosome, or nucleus. In some cases, a cellular fragment is amitochondria, rough endoplasmic reticulum, ribosome, smooth endoplasmicreticulum, chloroplast, golgi apparatus, golgi body, glycoprotein,glycolipid, cisternae, liposome, peroxisome, glyoxysome, centriole,cytoskeleton, lysosome, cilia, flagellum, contractile vacuole, vesicle,nuclear envelope, vacuole, cell membrane, microtubule, nucleolus, plasmamembrane, or chromatin.

One or more particles described herein can be in a sample. In somecases, one or more different types of particles described herein can bein a sample.

E. Particle Sizes

In some cases, a particle processed (e.g., chemically processed ortreated), purified, isolated, and/or concentrated using methods,compositions, devices, systems, and/or kits described herein has apredetermined particle size (or critical particle size). In some cases,particles with a size at least that of a predetermined particle size aredirected to a first outlet in a device, whereas particles less than apredetermined are directed to a second outlet in a device. In somecases, particle size is a diameter of a particle.

In some cases, a particle size is at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5,8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15,15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 μm.

In some cases, a particle size is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5,8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15,15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 μm.

In some cases, a particle size is less than 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7,7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5,15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100 μm, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 μm.

In some cases, a particle size is about 0.1 to about 1 μm, about 1 toabout 5 μm, about 5 to about 10 μm, about 10 to about 15 μm, about 10 toabout 20 μm, about 10 to about 25 μm, about 15 to about 25 μm, about 20to about 30 μm, about 30 to about 40 μm, about 40 to about 50 μm, about50 to about 60 μm, about 60 to about 70 μm, about 70 to about 80 μm,about 80 to about 90 μm, or about 90 to about 100 μm, about 100 to about200 μm, about 200 to about 300 μm, about 300 to about 400 μm, about 400to about 500 μm, about 500 to about 600 μm, or about 500 to about 1000μm.

In some cases, where a particle is polynucleotide, the polynucleotidecomprises at least 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150,200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, 2000,3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 20,000, 30,000,40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or 100,000 bases.

In some cases, where a particle is a polynucleotide, the polynucleotidecomprises about 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200,250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, 2000,3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 20,000, 30,000,40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 500,000,1,000,000, 5,000,000, or 10,000,000 bases. In some cases, apolynucleotide is a whole chromosome. In some cases, a polynucleotide isa human chromosome 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, X or Y.

In some cases, where a particle is a polynucleotide, the polynucleotidecomprises less than 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150,200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, 2000,3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 20,000, 30,000,40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 500,000,1,000,000, 5,000,000, or 10,000,000 bases.

In some cases, a polynucleotide comprises about 10 to about 100 bases,about 50 to about 100 bases, about 100 to about 200 bases, about 500 toabout 1000 bases, or about 1000 to about 2000 bases, about 2000 to about5000 bases, about 5000 to about 10,000 bases, about 10,000 to about50,000 bases, or about 50,000 to about 100,000 bases.

III. Samples

Particles from samples can be processed (e.g., chemically orenzymatically processed or treated), purified, isolated, and/orconcentrated using the methods, compositions, devices, systems, and/orkits described herein.

A. Types of Samples

In some cases, a sample is a biological sample. In some case, thebiological sample is blood. The blood sample can be, e.g., peripheralblood, maternal blood, G-CSF mobilized adult peripheral blood, or cordblood. Cord blood can be, e.g., umbilical cord blood, or placental cordblood.

In some cases, a biological sample is serum, plasma, sweat, tears, earflow, sputum, synovial fluid, lymph, bone marrow suspension, urine,saliva, semen, vaginal flow or secretion, cerebrospinal fluid, feces,cervical lavage, sebum, semen, prostatic fluid, Cowper's fluid,pre-ejaculatory fluid, female ejaculate, brain fluid (e.g.,cerebrospinal fluid), ascites, milk (e.g., breast milk), cerumen,secretions of the respiratory, intestinal or genitourinary tract,broncheoalveolar lavage fluid, amniotic fluid, aqueous humor, and watersamples). A sample can be fluids into which cells have been introduced(for example, culture media and liquefied tissue samples). A sample canbe a lysate. A biological sample can be hair, cyst fluid, pleural fluid,peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile,interstitial fluid, menses, pus, sebum, mucosal secretion, stool water,pancreatic juice, lavage fluid from sinus cavities, bronchopulmonaryaspirate, or blastocyl cavity fluid. A biological sample can be a tissuesample or biopsy. A fluid sample from an organism or one that has beensolubilized can be at least 1, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, 10, 20, 50,75, 100, 200, 500, 1000 or 1500 mL.

In some cases, a biological sample is from an animal, e.g., human,mouse, rat, cat, dog, cow, chicken, donkey, rabbit, chimpanzee, gorilla,orangutan, horse, guinea pig, pig, or rhesus monkey.

In some cases, a biological sample is from a plant. In some cases, abiological sample comprises a plant.

In some cases, a biological sample is from a fugus. In some cases, abiological sample comprises a fungus.

In some cases, a sample comprises leukocytes and erythrocytes.

In some cases, a sample comprises cells. In some cases, a samplecomprises dead cells, and/or debris. The methods, compositions, devices,systems, and/or kits described herein can be used for size-based removalof debris and/or dead cells from a sample comprising cells. In somecases, the methods, compositions, devices, systems, and/or kitsdescribed herein can be used for cell wash procedures. In some cases,the sample is a cell culture sample. In some cases, the methods,compositions, devices, systems, and/or kits described herein can be usedto process (e.g., chemically and/or enzymatically process or treat),isolate, purify, and/or concentrate cells from other components in acell culture sample, e.g, medium, growth factors, etc.

In some cases, a sample comprises a buffer. The buffer can be free orsubstantially free of a reagent. In some cases, the methods,compositions, devices, systems, and/or kits described herein can be usedfor buffer/medium exchange.

In some cases, a sample comprises enzyme digested adipose tissue. Insome cases, the enzyme digested adipose tissue is a source forautologous progenitor cells. In some cases, the methods, compositions,devices, systems, and/or kits described herein can be used to clean-upenzyme (e.g. collagenase) digested adipose tissue as a source forautologous progenitor cells, e.g., purify stem cells from the enzymedigested adipose tissue.

In some cases, a sample comprises cancer cells from tumors. In somecases, the methods, compositions, devices, systems, and/or kitsdescribed herein can be used to process (e.g., chemically process ortreat), isolate, purify, and/or concentrate cancer cells from tumors.

In some cases, a sample comprises infiltrating or stromal host cellsfrom a tumor. In some cases, the methods, compositions, devices,systems, and/or kits described herein can be used to process (e.g.,chemically and/or enzymatically process or treat), isolate, purify,and/or concentrate infiltrating cells or stromal host cells from atumor. For example, tumor-infiltrating lymphocytes can be white bloodcells that have left the bloodstream and migrated to a tumor. Stromalcells can be connective tissue. Stromal cells can provide anextracellular matrix on which tumors can grow.

In some cases, a sample is an industrial sample. In some cases, themethods, compositions, devices, systems, and/or kits described hereincan be used to process (e.g., chemically and/or enzymatically process ortreat), isolate, purify, and/or concentrate particles in an industrialsample.

In some cases, a sample comprises algae, yeast, bacteria, or a virus. Insome cases, methods, compositions, devices, systems, and/or kitsdescribed herein can be used to process (e.g., chemically and/orenzymatically process or treat), isolate, purify, and/or concentratealgae, yeast, bacteria, and/or a virus. For example, a sample with yeastcan be a beer production sample. Methods, compositions, devices,systems, and/or kits described herein can be used to process (e.g.,chemically and/or enzymatically process or treat), isolate, purify,and/or concentrate yeast from the sample from a beer production sample.

In some cases, a sample comprises an antibody. In some cases, methods,compositions, devices, systems, and/or kits described herein can be usedto process (e.g., chemically and/or enzymatically process or treat),isolate, purify, and/or concentrate and antibody from a samplecomprising an antibody.

In some cases, a sample comprises plants, mitochondria, lentivirus,exosomes, or dividing cells. Methods, compositions, devices, systems,and/or kits described herein can be used to process (e.g., chemicallyand/or enzymatically process or treat), isolate, purify, and/orconcentrate plants, mitochondria, lentivirus, exosomes, or dividingcells from the sample.

In some cases, a sample comprises cells at different stages in the cellcycle, G0 (Gap 0/Resting), G1 (Gap 1), S (Synthesis), M (Mitosis), or G2(Gap 2). Cells can have different sizes at different stages of the cellcycle. In some cases, the methods and devices described herein are usedto separate cells at different stages of the cell cycle.

In some cases, a sample is from a body of water. A body of water can be,e.g., from a creek, pond, river, ocean, lake, sea, puddle, stream canal,wetland, marsh, reservoir, harbor, bay, artificial lake, barachois,basin, bayou, beck, bight, billabong, boil, brook, burn, channel, cove,draw, estuary, firth, fjord, glacier, gulf, inlet, kettle, kill, lagoon,loch, mangrove swamp, Mediterranean sea, mere, mill pond, moat, oxbowlake, phytotelma, pool (swimming pool, reflecting pool), pothole, rapid,roadstead, run, salt marsh, sea loch, sea lough, source, spring, strait,stream, subglacial, lake, swamp, tarn, tide pool, vernal pool, wash, orwetland.

In some cases, a sample is from a bioterror attack. In some cases, asample from a bioterror attack comprises a virus, e.g., smallpox virusor influenza virus. In some cases, a sample from a bioterror attackcomprises anthrax. In some cases, a sample from a bioterror attackcomprises more than one type of infective agent.

In some cases, the methods described herein are used purify viruses fromcells, e.g., a lentivirus. Examples of lentivirus include humanimmunodeficiency virus (HIV), simian immunodeficiency virus, felineimmunodeficiency virus, puma lentivirus, equine infectious anemia virus,bovine immunodeficiency virus, caprine arthritis encephalitis virus, orVisna virus. In some cases, a virus can be purified away from cellsand/or cellular debris.

In some cases, a sample is from a hospital or other medical health carefacility. In some cases, a sample is from a wastewater treatmentfacility. In some cases, a sample is from an algal biofuel productionfacility. In some cases, a sample is from a brewery. In some cases, asample is from a public water system. In some cases, a sample is from asewage system.

In some cases, a sample is from a chemical spill. In some cases, asample is from a mine, e.g., coal mine. In some cases, a sample is anarcheological sample. In some cases, a sample is a forensic sample.

In some cases, the erythrocytes in samples are not lysed. In some cases,erythrocytes in samples are lysed.

In some cases, a sample comprises one or more labels. In some cases, asample comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 different labels. In some cases, a label is anantibody, antibody fragment, dye, stain (e.g., ethidium bromide),nucleic acid adapter, radioactive particle, fluorescent particle,oligonucleotide, probe, or fluorescently-labeled probe. In some cases, alabel is bound, linked, or conjugated to an antibody, antibody fragment,dye, stain (e.g., ethidium bromide), nucleic acid adapter, radioactiveparticle, fluorescent particle, oligonucleotide, probe, orfluorescently-labeled probe. In some cases, a device, method,composition, system, and/or kit as provided herein process a particle(e.g., cell) such that the particle (e.g., cell) comprises a first labeland a second label. The first and second label can be different labels.The first label can be bound, conjugated, or linked to a binding agentas provided herein directed to a marker of the surface of the particle(e.g. cell surface marker), while the second label can be bound,conjugated, or linked to binding agent as provided herein directed to amarker in the interior of the particle (e.g., intracellular marker). Thelabel can be any label as provided herein.

In some cases, a sample comprises an enzyme, e.g., a restriction enzyme,kinase (e.g., DNA kinase (e.g., T4 polynucleotide kinase), proteinkinase, e.g., serine kinase, threonine kinase, tyrosine kinase), DNase,RNase, phosphatase, ligase (e.g., RNA ligase, DNA ligase), horseradishperoxidase (HRP), alkaline phosphatase (AP), glucose oxidase, polymerase(e.g., DNA polymerase (e.g., thermostable DNA polymerase, Taqpolymerase) RNA polymerase), terminal deoxynucleotidyl transferase,reverse transcriptase (e.g., viral reverse-transcriptase, non-viralreverse transcriptase), telomerase, methylase, or topoisomerase. In somecases, methods and/or device used herein can be used to separate a labelor enzyme from another component of a sample, e.g., a polynucleotide orcell.

B. Number of Particles/Numbers of Different Types of Particles in aSample

A sample can comprise one or more first particles. In some cases, asample can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100,500, 1000, 10,000, 100,000, 1,000,000, 10,000,000, 100,000,000,1,000,000,000, 10,000,000,000, 100,000,000,000, or 1,000,000,000,000first particles. A sample can comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 50, 100, 500, 1000, 10,000, 100,000, 1,000,000, 10,000,000,100,000,000, 1,000,000,000, 10,000,000,000, 100,000,000,000, or1,000,000,000,000 first particles. In some cases, a sample comprisesabout 10 to about 100 first particles, about 5 to about 10 firstparticles, about 10 to about 50 first particles, about 50 to about 100first particles, about 100 to about 1000 first particles, about 1000 toabout 10,000 first particles, about 10,000 to about 100,000 firstparticles, about 100,000 to about 1,000,000 first particles, about1,000,000 to about 10,000,000 first particles, about 10,000,000 to about100,000,000 first particles, about 100,000,000 to about 1,000,000,000first particles, about 1,000,000,000 to about 10,000,000,000 firstparticles, about 10,000,000,000 to about 100,000,000,000 firstparticles, or about 100,000,000,000 to about 1,000,000,000,000 firstparticles.

In some cases, a sample comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 50, 100, 500, 1000, 10,000, 100,000, 1,000,000, 10,000,000,100,000,000, 1,000,000,000, 10,000,000,000, 100,000,000,000, or1,000,000,000,000 total particles. A sample can comprise about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 50, 100, 500, 1000, 10,000, 100,000, 1,000,000,10,000,000, 100,000,000, 1,000,000,000, 10,000,000,000, 100,000,000,000,or 1,000,000,000,000 first particles, In some cases, a sample comprisesabout 10 to about 100 total particles, about 5 to about 10 totalparticles, about 10 to about 50 total particles, about 50 to about 100total particles, or about 100 to about 1000 total particles, about 1000to about 10,000 total particles, about 10,000 to about 100,000 totalparticles, about 100,000 to about 1,000,000 total particles, about1,000,000 to about 10,000,000 total particles, about 10,000,000 to about100,000,000 total particles, about 100,000,000 to about 1,000,000,000total particles, about 1,000,000,000 to about 10,000,000,000 totalparticles, about 10,000,000,000 to about 100,000,000,000 totalparticles, or about 100,000,000,000 to about 1,000,000,000,000 totalparticles.

A sample can comprise one or more different types of particles. A samplecan comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 500, 1000,10,000, 100,000, 1,000,000, 10,000,000, 100,000,000, 1,000,000,000,10,000,000,000, 100,000,000,000, or 1,000,000,000,000 different types ofparticles. A sample can comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,50, 100, 500, 1000, 10,000, 100,000, 1,000,000, 10,000,000, 100,000,000,1,000,000,000, 10,000,000,000, 100,000,000,000, or 1,000,000,000,000different types of particles. In some cases, a sample comprises about 10to about 100 different types of particles, about 5 to about 10 differenttypes of particles, about 10 to about 50 different types of particles,about 50 to about 100 different types of particles, or about 100 toabout 1000 different types of particles, about 1000 to about 10,000different types of particles, about 10,000 to about 100,000 differenttypes of particles, about 100,000 to about 1,000,000 different types ofparticles, about 1,000,000 to about 10,000,000 different types ofparticles, about 10,000,000 to about 100,000,000 different types ofparticles, about 100,000,000 to about 1,000,000,000 different types ofparticles, about 1,000,000,000 to about 10,000,000,000 different typesof particles, about 10,000,000,000 to about 100,000,000,000 differenttypes of particles, or about 100,000,000,000 to about 1,000,000,000,000different types of particles.

C. Ratio of First and Second Particles in a Sample

In some cases, a sample comprises a first particle and a secondparticle. In some cases, the ratio of the abundance of the firstparticle to the second particle in the sample is less than 1:1, 1:10,1:100, 1:1000, 1:10,000, 1:100,000, 1:1,000,000, 1:10,000,000,1:100,000,000, or 1:1,000,000,000. In some cases, the ratio of theabundance of the first particle to the second particle in the sample isgreater than 1:1, 1:10, 1:100, 1:1000, 1:10,000, 1:100,000, 1:1,000,000,1:10,000,000, 1:100,000,000, or 1:1,000,000,000. In some cases, theratio of the abundance of the first particle to the second particle inthe sample is about 1:1, 1:10, 1:100, 1:1000, 1:10,000, 1:100,000, or1:1,000,000, 1:10,000,000, 1:100,000,000, or 1:1,000,000,000.

In some cases, a sample comprises a rare cell type. In some cases, theratio of the abundance of the rare cell type to the abundance of cellsof one or more other cell types in a sample is about 1:100, 1:1000,1:10,000, 1:100,000, 1:1,000,000, 1:10,000,000, 1:100,000,000, or1:1,000,000,000. In some cases the ratio of abundance of cells of therare cell type to the abundance of cells of one or more other cell typesis less than 1:100, 1:1000, 1:10,000, 1:100,000, 1:1,000,000,1:10,000,000, 1:100,000,000, or 1:1,000,000,000.

D. Sample Dilution

In some cases, a sample is diluted. In some cases, a sample, e.g., ablood sample, is diluted before it is applied to a device describedherein. A sample can be diluted, e.g., in order to prevent clogging of adevice described herein. In some cases, a sample is diluted after beingpassed through a device described herein.

A sample can be diluted at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9,9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16,16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700,1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900,or 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000-fold.

In some cases, a sample is diluted about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5,8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15,15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500,1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700,2800, 2900, or 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000-fold.

A sample can be diluted, e.g., by adding water, buffer, and/or otherfluid to the sample. In some cases, a sample is diluted by adding anadditive.

E. Clogging and Sample Additives

Disclosed herein are methods for processing large volumes of blood withdeterministic lateral displacement arrays, e.g., for isolation of rarecells. In some cases, the disclosed methods can be used to extractcirculating tumor cells from large volumes (˜100 mL) of whole blood ofcancer patients, or, e.g, to extract stem cells from large volumes ofumbilical cord blood. The disclosed methods are also useful for generalprocessing of blood using DLD arrays.

In some cases, deterministic lateral displacement (DLD) arrays are usedto extract rare cells from hundreds of microliters of blood. Using thedisclosed methods, DLD arrays can be used to extract rare cells fromhundreds of milliliters of blood. The “robustness” of the techniqueagainst clogging and fouling for lower throughputs for other bloodapplications is also improved.

In some cases, a process for reducing clogging comprises a combinationof four techniques: 1) increase in the concentration of thecalcium-chelating anti-coagulant EDTA from, e.g., 1 mM to, e.g, 5 mM; 2)add a direct thrombin inhibitor, e.g., PPACK at a concentration of,e.g., 40 μm; 3) increase in the flow velocity 10×; and a 3× increase inthe dilution of blood. In some cases, only one technique is performed.In some cases, two or more of the techniques are performed.

In some cases, a kit is provided comprising a calcium-chelating agentand a thrombin inhibitor. In some cases, the kit comprises EDTA andPPACK.

Using blood with fluorescently stained leukocytes, the level of cloggingcan be measured as a function of the volume of blood that had passedthrough the DLD array. Employing the disclosed approach, ˜100 mL ofblood can pass through the DLD array before clogging using a combinationof the four methods above, compared to a few hundred microliterspreviously.

The combination of EDTA and PPACK can be used as a running buffer fordilution of the blood in preparation for processing of the blood withDLD arrays.

The references listed herein are also part of the application and areincorporated by reference in their entirety as if fully set forthherein.

Deterministic lateral displacement arrays can be used to captureleukocytes and circulating tumor cells from blood with high enrichmentand at high flow rates.

In some cases, the volume of blood that can be processed with thesedevices is limited due to clogging of the micro-post (obstacle) array.In some cases, by removing platelets from blood before putting the bloodin the arrays, platelets can be identified as a dominant contributor toclogging. For example, running leukocytes alone can lead to far lessclogging than running blood. In some cases, a biological mechanismcausing clogging can be disabled, which can yield at least a 40-foldreduction in clogging. In some cases, higher flow rates and greaterdilution of blood can be used to achieve a further reduction in cloggingof a micro-post array.

The physiological conditions in devices comprising an array of obstacles(high shear, rapid repeated acceleration and deceleration) can bedifferent from those found in typical situations involving bloodclotting studies.

Clogging of a micro-post (obstacle) array can be caused by one or bothof two complementary, mutually dependent processes involved inhemostasis: coagulation and platelet activation, which lead to a clot.In some cases, clots (which then can trap leukocytes) can cause theclogging.

FIG. 35 illustrates a simplified view of possible ways in which thesetwo processes can interact to cause clogging as well as underlyingmechanisms which can be attacked to disable these mechanisms. The cyclemay be initiated by mechanical stress on the platelets from shear forcesas the platelets pass between the posts in a micro-post array, or theirrapid acceleration and deceleration caused by a micro-array structure.

Coagulation-related processes are on the left side of the diagram, andthe platelet processes are on the right side of the diagram. They caninter-relate through thrombin and calcium pathways and dependencies. Insome cases, both the thrombin and the calcium pathways/dependencies canbe addressed for maximum effectiveness.

In some cases, high flow rate and dilution both lead to an increase inthe maximum throughput of whole blood before significant clogging canoccur.

In some cases, a dominant clogging mechanism is the activity ofcalcium-dependent integrins on platelet surfaces, the interaction ofwhich can lead to aggregation of platelets. In some cases,calcium-dependent integrins are one of the dominant contributors toplatelet-induced clogging. In some cases, increasing the concentrationof EDTA from 1 mM to 5 mM can result in an 8-fold reduction in clogging.In some cases, acid citrate dextrose (ACD), like EDTA, chelates calciumin blood plasma, has a similar effect. The chelation of calcium can alsoreduce the coagulation pathways (on the left of the diagram).

In some cases, a dominant clogging mechanism is due to thrombin effects,e.g., thrombin-induced platelet activation. Thrombin can catalyze theconversion of fibrinogen into fibrin as a part of a coagulation cascade.In some cases, thrombin is a potent platelet-activating agonist. Heparincan be effective in reducing clogging—it can reduce the formation ofthrombin.

In some cases, a calcium chelator can be combined with a thrombininhibitor. In some cases, inhibiting thrombin with the direct thrombininhibitor PPACK can achieve a further 5-fold reduction in clogging ontop of that achieved with a 5 mM concentration of EDTA.

FIG. 36 shows these results for the case of an array with 40 umtriangular posts with 27 um gaps for leukocyte separation from blood.

Deterministic lateral displacement (DLD) arrays have been used toconcentrate circulating tumor cells (CTCs) in diluted whole blood atflow rates as high as 10 mL/min with capture efficiencies exceeding 85%(K. Loutherback et al., AIP Advances, 2012). In some cases, theequivalent volume of undiluted blood that can be processed is limited to0.3 mL per DLD array due to clogging of the array. Since theconcentration of CTCs can be as low as 1-10 cells/mL in clinicalsamples, increasing the volume of blood that can be processed with DLDarrays can allow for collection of sufficient numbers of CTCs forbiological experiments or clinical diagnostic studies. Furthermore, bybumping large cells, such as CTCs, into a buffer stream, DLD arrays canbe used to harvest CTCs free of the background of smaller particles,such as leukocytes, erythrocytes, and platelets present in blood or,free of plasma, resulting in a highly enriched or concentrated product(see e.g., J. A. Davis, et al., PNAS, 2006).

In some cases, two biological mechanisms can cause clogging of thearray, and these two mechanisms can be inhibited. In some cases,shear-induced platelet aggregation is only a minor contributor toclogging of the array. In some cases, by comparing the reduction inclogging achieved by the calcium-chelating anticoagulants EDTA and ACDto that achieved by the indirect thrombin inhibitor heparin as well asby measuring the EDTA concentration-dependent reduction in clogging,activity of calcium-dependent integrins as a dominant contributor toclogging can be identified. In some cases, combining EDTA with thedirect thrombin inhibitor PPACK can be used to identify thrombin-inducedplatelet activation as the second dominant mechanism contributing toclogging. Using a combination of EDTA and PPACK, a 40-fold decrease inclogging of the array can be demonstrated, which can allow acommensurate increase in the volume of blood processed. Based on data ina single-channel device (2 mm wide), we can expect a complete chip to beable to process >100 mL quantities of blood in 30 minutes withoutsignificant clogging. Finally, in some cases, the glycoprotein 11b/Illaintegrin complex, which is activated by shear forces, can be inhibitedusing the glycoprotein 11b/Illa inhibitor tirofiban to show thatshear-induced platelet aggregation plays only a minor role in cloggingof the array.

In some cases, a sample can comprise one or more additives. In somecases, a chelating agent is added to a sample. In some cases, thechelating agent comprises a calcium-chelating agent. In some cases, thechelating agent comprises acetylacetone, aerobactin,aminoethylethanolamine, aminopolycarboxylic acid, ATMP, BAPTA, BDTH2,benzotriazole, Bipyridine, 2,2′-Bipyridine, 4,4′-Bipyridine,1,2-Bis(dimethylarsino)benzene, 1,2-Bis(dimethylphosphino)ethane,1,2-Bis(diphenylphosphino)ethane, Catechol, Chelex 100, Citric acid,Corrole, Crown ether, 18-Crown-6, Cryptand, 2.2.2-Cryptand, Cyclen,Deferasirox, Deferiprone, Deferoxamine, Dexrazoxane,Trans-1,2-Diaminocyclohexane, 1,2-Diaminopropane, Dibenzoylmethane,Diethylenetriamine, Diglyme, 2,3-Dihydroxybenzoic acid, Dimercaprol,2,3-Dimercapto-1-propanesulfonic acid, Dimercaptosuccinic acid,Dimethylglyoxime, DIOP, Diphenylethylenediamine, DOTA, DOTA-TATE, DTPMP,EDDH, EDDS, EDTMP, EGTA, 1,2-Ethanedithiol, Ethylenediamine,Ethylenediaminetetraacetic acid (EDTA), Etidronic acid, Extendedporphyrins, Ferrichrome, Fluo-4, Fura-2, Gluconic acid,Glyoxal-bis(mesitylimine), Hexafluoroacetylacetone, Homocitric acid,Iminodiacetic acid, Indo-1, Metal acetylacetonates, Metal dithiolenecomplex, Metallacrown, Nitrilotriacetic acid, Pendetide, Penicillamine,Pentetic acid, Phanephos, Phenanthroline, O-Phenylenediamine,Phosphonate, Phytochelatin, Polyaspartic acid, Porphin, Porphyrin,3-Pyridylnicotinamide, 4-Pyridylnicotinamide, Sodiumdiethyldithiocarbamate, Sodium polyaspartate, Terpyridine,Tetramethylethylenediamine, Tetraphenylporphyrin,1,4,7-Triazacyclononane, Triethylenetetramine, Triphos, Trisodiumcitrate, or 1,4,7-Trithiacyclononane.

In some cases, the sample, e.g., a blood sample, is collected in a tubecomprising K₂EDTA or K₃EDTA.

In some cases, the sample comprises an agent that reduces the activityof calcium-dependent integrins. In some cases, the sample comprises anagent that reduces calcium dependent thrombin formation.

In some cases, an agent that chelates calcium comprises acid citratedextrose (ACD). The final concentration of ACD in a sample, e.g., ablood sample, can be 10%.

In some cases, the chelating agent is EDTA. In some cases, the calciumchelating agent is EDTA. In some cases, the final concentration of thechelating agent in the sample is at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5,8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15,15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 mM. In some cases,the final concentration of EDTA in the sample is at least 0.1, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6,6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14,14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23,24, or 25 mM. In some cases, the concentration of EDTA is about 2 mM toabout 7 mM, or about 3 mM to about 6 mM.

In some cases, one or more thrombin inhibitors are added to a sample,e.g., a blood sample. In some cases, a thrombin inhibitor is a directthrombin inhibitor. In some cases, a direct thrombin inhibitor is abivalent thrombin inhibitor. In some cases, a direct thrombin inhibitoris a univalent thrombin inhibitor. In some cases, a direct thrombininhibitor is an allosteric inhibitor. A bivalent direct thrombininhibitor can be hirudin, bivalirudin, lepirudin, or desirudin. Aunivalent direct thrombin inhibitor can be argatroban, melagatran,ximelagatran, or dabigatran. An allosteric direct thrombin inhibitor canbe a DNA aptamer, benzofuran dimer, benzofuran trimer, or polymericlignin. In some cases, a direct thrombin inhibitor is PPACK(D-Phe-Pro-Arg-CMK).

In some cases, the thrombin inhibitor is PPACK (D-Phe-Pro-Arg-CMK),benzamidine hydrochloride, p-APMSF, p-APMSF hydrochloride, TLCKhydrochloride, uPA inhibitor, PPACK dihydrochloride, or PPACKdihydrochloride biotinylated. In some cases, Heparin is a thrombininhibitor.

In some cases, the final concentration of the thrombin inhibitor, e.g.,direct thrombin inhibitor in a sample is at least 1, 1.5, 2, 2.5, 3,3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5,12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5,19, 19.5, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200,250, 300, 350, or 400 μM. In some cases, a final concentration athrombin inhibitor in a sample is about 30 to about 50 μM, or about 20to about 60 μM.

In some cases, the final concentration of PPACK in a sample is at least1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5,10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5,17, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,125, 150, 175, 200, 250, 300, 350, or 400 μM. In some cases, a finalconcentration of PPACK in a sample is about 30 to about 50 μM, or about20 to about 60 μM.

In some cases, a chelating agent and a thrombin inhibitor are added to asample. In some cases, a calcium chelating agent and a thrombininhibitor are added to a sample. In some cases, a chelating agent and athrombin inhibitor are added to a sample, and the sample is diluted atleast 3 fold.

In some cases, a sample comprises EDTA and PPACK. In some cases, asample comprises EDTA at a concentration of about 5 mM and PPACK at aconcentration of about 40 μM. In some cases, a blood sample comprisesEDTA at a concentration of about 5 mM and PPACK at a concentration ofabout 40 μM

In some cases, a blood sample is diluted about 3 fold, and the dilutedblood sample comprises EDTA and PPACK. In some cases, a blood sample isdiluted about 3 fold, and the diluted blood sample comprises EDTA at aconcentration of about 5 mM and PPACK at a concentration of about 40 μM.

In some cases, a sample, e.g., a blood sample, comprises one or moreadditives, e.g., sodium fluoride (NaF), Heparin, EDTA, or sodiumcitrate. In some cases, an additive is an anticoagulant or antiplateletagent, e.g., clopidogrel, prasugrel, ticagrelor, ticlopidine,argatroban, bivalirudin, dalteparin, enoxaparin, fondaparinux, heparin,heparin lock flush, lepirudin, anagrelide, apixaban, aspirin,aspirin/dipyridamole, cilostazol, dabigatran, dipyridamole, batroxobin,hementin, rivaroxaban, warfarin, or urokinase. In some cases, ananticoagulant is an antithrombic, fibrinolytic, or thrombolytic.

In some cases, whole blood is diluted with 1×PBS with 2 mM EDTA and 0.5%BSA.

F. Sample Volumes

Samples can be applied to devices described herein, e.g., devices withordered arrays of obstacles, e.g., deterministic lateral displacement(DLD) devices. The volume of sample that can be applied to a deviceand/or processed by a device can be at least 0.01, 0.02, 0.03, 0.04,0.05, 0.06, 0.7, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9,9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16,16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700,1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900,or 3000 mL.

The volume of sample that can be applied to a device and/or processed bya device can be less than 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.7, 0.08,0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3,3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5,12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5,19, 19.5, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000,2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, or 3000 mL.

The volume of sample that can be applied to a device and/or processed bya device can be about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.7, 0.08,0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3,3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5,12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5,19, 19.5, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000,2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, or 3000 mL.

The volume of sample that can be applied to device and/or processed by adevice can be about 0.1 to about 1 mL, about 1 to about 10 mL, about 10mL to about 20 mL, about 10 mL to about 50 mL, about 10 mL to about 100mL, about 20 mL to about 100 mL, about 100 mL to about 300 mL, about 100mL to about 1000 mL, about 100 mL to about 500 mL, or about 100 mL toabout 3000 mL.

G. Concentration of Particles in a Sample

In some cases, a concentration of particles in a sample is about 1, 5,10, 50, 100, 500, 1000, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, or 10¹¹ permL of sample.

In some cases, a concentration of particle in a sample is less than 1,5, 10, 50, 100, 500, 1000, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, or 10¹¹per mL of sample.

In some cases, a concentration of particles in a sample is at least 1,5, 10, 50, 100, 500, 1000, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, or 10¹¹per mL of sample.

IV. Devices

Exemplary devices for separating particles based on size are described,e.g., in U.S. Pat. Nos. 7,150,812, 7,318,902, 7,472,794, 7,735,652,7,988,840, 8,021,614, 8,282,799, 8,304,230, 8,579,117, and PCTPublication No. WO2012094642, which are herein incorporated by referencein their entireties. Particles and samples described herein can beapplied to devices described herein for size-based separation, e.g.,high throughput size based separation.

The disclosure relates generally to the field of separation of particlessuch as spheres, cells, viruses, and molecules. The disclosure relatesto separation of particles based on their flow behavior in afluid-filled field of obstacles in which advective transport ofparticles by a moving fluid overwhelms the effects of diffusive particletransport.

Separation of particles by size or mass can be a fundamental analyticaland preparative technique in biology, medicine, chemistry, and industry.Conventional methods include gel electrophoresis, field-flowfractionation, sedimentation and size exclusion chromatography. Morerecently, separation of particles and charged biopolymers has beendescribed using arrays of obstacles through particles pass under theinfluence of fluid flow or an applied electrical field. Separation ofparticles by these obstacle-array devices can be mediated byinteractions among the biopolymers and the obstacles and by the flowbehavior of fluid passing between the obstacles.

A variety of microfabricated sieving matrices have been disclosed forseparating particles (Chou et. al., 1999, Proc. Natl. Acad. Sci.96:13762; Han, et al., 2000, Science 288:1026; Huang et al., 2002, Nat.Biotechnol. 20:1048; Turner et al., 2002, Phys. Rev. Lett.88(12):128103; Huang et al., 2002, Phys. Rev. Lett. 89:178301; U.S. Pat.No. 5,427,663; U.S. Pat. No. 7,150,812; U.S. Pat. No. 6,881,317). Thesematrices can depend on accurate fabrication of small features (e.g.,posts, or obstacles in a microfluidic channel). The accuracy with whichsmall features can be fabricated can be limited in all micro-fabricationmethods, especially as feature size decreases. The strength and rigidityof materials in which small features of fabricated can also limit thepractical usefulness of the fabricated device. Furthermore, the smallsize of the gaps between obstacles in such matrices can render thematrices susceptible to clogging by particles too large to fit betweenthe obstacles. Micrometer- and nanometer-scale manufacturing can alsorequire state-of-the-art fabrication techniques, and devices fabricatedusing such methods can have high cost.

Bump array (also known as “obstacle array”) devices have been described,and their basic operation is explained, for example in U.S. Pat. No.7,150,812, which is incorporated herein by reference in its entirety.Referring to FIGS. 3 and 4 of U.S. Pat. No. 7,150,812, a bump array canoperate essentially by segregating particles passing through an array(generally, a periodically-ordered array) of obstacles, with segregationoccurring between particles that follow an “array direction” that isoffset from the direction of bulk fluid flow or from the direction of anapplied field.

At the level of flow between two adjacent obstacles under conditions ofrelatively low Reynold's number, fluid flow can occur in a laminarfashion. Considering the volumetric flow between two obstacles inhypothetical layers (e.g., modeling the flow by considering multipleadjacent stream tubes of equal volumetric flow between the obstacles, asshown in FIG. 8 of U.S. Pat. No. 7,150,812), the likelihood that fluidin a layer will pass on one side or the other of the next (i.e.,downstream) obstacle can be calculable by standard methods (see, e.g.,Inglis et al., 2006, Lab Chip 6:655-658). For an ordered array ofobstacles offset from the direction of bulk fluid flow, the arrangementof the obstacles can define an array direction corresponding to thedirection in which the majority of fluid layers between two obstaclestravels. A minority of fluid layers can travel around the downstreamobstacle in a direction other than the array direction.

The path that a particle passing between the two obstacles can take candepend on the flow of the fluid in the layers occupied by the particle.Conceptually, for a particle having a size equal to one of thehypothetical fluid layers described in the preceding paragraph, theparticle can follow the path of the fluid layer in which it occurs,unless it diffuses to a different layer. For particles larger than asingle fluid layer, the particle can take the path corresponding to themajority of the fluid layers acting upon it. Particles having a sizegreater than twice the sum of the thicknesses of the minority of layersthat travel around a downstream obstacle in the direction other than thearray direction can be acted upon by more fluid layers moving in thearray direction, meaning that such particles will travel in the arraydirection. This concept is also illustrated in FIGS. 5-11 of U.S. Pat.No. 7,150,812. Thus, there can be a “critical size” for particlespassing between two obstacles in such an array, such that particleshaving a size greater to that critical size can travel in the arraydirection, rather than in the direction of bulk fluid flow and particleshaving a size less than the critical size can travel in the direction ofbulk fluid flow. Particles having a size precisely equal to the criticalsize can have an equal chance of flowing in either of the twodirections. By operating such a device at a high Peclet number (i.e.,such that advective particle transport by fluid layers greatly outweighsdiffusive particle between layers), the effects of diffusion ofparticles between fluid layers can be ignored.

A. Bump Arrays

Described herein are ways of structuring and operating obstacle arraysfor separating particles. In some obstacle arrays, obstacles have shapesand are arranged such that the profile of fluid flow through gapsbetween adjacent obstacles is symmetrical about the center line of thegap. The geometry of the adjacent obstacles can be such that theportions of the obstacles defining the gap are symmetrical about theaxis of the gap that extends in the direction of bulk fluid flow. Thevelocity or volumetric profile of fluid flow through such gaps can beapproximately parabolic across the gap, with fluid velocity and fluxbeing zero at the surface of each obstacle defining the gap (assumingno-slip flow conditions) and reaches a maximum value at the center pointof the gap. The profile being parabolic, a fluid layer of a given widthadjacent to one of the obstacles defining the gap can contain an equalproportion of fluid flux as a fluid layer of the same width adjacent theother obstacle that defines the gap meaning that the critical size ofparticles that are ‘bumped’ during passage through the gap is equalregardless of which obstacle the particle travels near.

In some cases, particle size-segregating performance of an obstaclearray can be improved by shaping and disposing the obstacles such thatthe portions of adjacent obstacles that deflect fluid flow into a gapbetween obstacles are not symmetrical about the axis of the gap thatextends in the direction of bulk fluid flow. Such lack of flow symmetryinto the gap can lead to a non-symmetrical fluid flow profile within thegap. Concentration of fluid flow toward one side of a gap (i.e., aconsequence of the non-symmetrical fluid flow profile through the gap)can reduce the critical size of particles that are induced to travel inthe array direction, rather than in the direction of bulk fluid flow.This can be so because the non-symmetry of the flow profile can causedifferences between the width of the flow layer adjacent to one obstaclethat contains a selected proportion of fluid flux through the gap andthe width of the flow layer that contains the same proportion of fluidflux and that is adjacent the other obstacle that defines the gap. Thedifferent widths of the fluid layers adjacent to obstacles define a gapthat exhibits two different critical particle sizes. A particletraversing the gap can be bumped (i.e., travel in the array direction,rather than the bulk fluid flow direction) if it exceeds the criticalsize of the fluid layer in which it is carried. Thus, it is possible fora particle traversing a gap having a non-symmetrical flow profile to bebumped if the particle travels in the fluid layer adjacent to oneobstacle, but to be not-bumped if it travels in the fluid layer adjacentto the other obstacle defining the gap.

Particles traversing an obstacle array pass through multiple gapsbetween obstacles, and have multiple opportunities to be bumped. When aparticle traverses a gap having a non-symmetrical flow profile, theparticle can be bumped if the size of the particle exceeds the(different) critical sizes defined by the flow layers adjacent to thetwo obstacles defining the gap. However, the particle can sometimes bebumped if the size of the particle exceeds the critical size defined bythe flow layer adjacent to one of the two obstacles, but does not exceedthe critical size defined by the flow layer adjacent to the otherobstacle. In some cases, particles that do not exceed the critical sizedefined by the flow layer adjacent to either of the obstacles cannot bebumped. There are at least two implications that follow from thisobservation.

First, in an obstacle array in which the obstacles define gaps having anon-symmetrical flow profile, particles having a size that exceeds thesmaller of the two critical sizes defined by the flow layers adjacent tothe obstacles can be separated from particles having a size smaller thanthat smaller critical size. The critical size defined by a gap can bedecreased by altering the symmetry of flow through the gap withoutnecessarily decreasing the size of the gap (“G” in FIG. 1). Decreasinggap size can increase the cost and difficulty of producing the array.Conversely, for a given critical size, the size of the gap defining thatcritical size can be increased by altering the symmetry of flow throughthe gap. Because smaller gaps are more likely to clog than larger ones,this arrangement can improve the operability of the arrays, allowinggreater throughput and lower likelihood of clogging.

Second, in an obstacle array in which the obstacles define gaps having anon-symmetrical flow profile, particles can be separated into threepopulations: i) particles having a size smaller than either of thecritical sizes defined by the flow layers adjacent to the obstacles; ii)particles having a size intermediate between the two critical sizesdefined by the flow layers adjacent to the obstacles; and iii) particleshaving a size larger than either of the critical sizes defined by theflow layers adjacent to the obstacles.

In another aspect, decreasing the roundness of edges of obstacles thatdefine gaps can improve the particle size-segregating performance of anobstacle array. By way of example, arrays of obstacles having atriangular cross-section with sharp vertices can exhibit a lowercritical particle size than do arrays of identically-sized and -spacedtriangular obstacles having rounded vertices.

Thus, by sharpening the edges of obstacles defining gaps in an obstaclearray, the critical size of particles deflected in the array directionunder the influence of bulk fluid flow can be decreased withoutnecessarily reducing the size of the obstacles. Conversely, obstacleshaving sharper edges can be spaced farther apart than, but still yieldparticle segregation properties equivalent to, identically-sizedobstacles having less sharp edges.

In yet another aspect, shaping the obstacles in an obstacle array insuch a way that the geometry of the obstacles encountered by fluidflowing through the array in one direction differs (and defines adifferent critical particle size) from the geometry of the obstaclesencountered by fluid flowing through the array in a second direction.For example, fluid flowing through the array illustrated in FIG. 1 in aleft-to-right direction encounters and flows around the rounded verticesof the right triangular posts of the array (in this flow direction, theprofile of fluid flow through the gaps is asymmetric about the axis ofthe gaps). However, fluid flowing through the same array in aright-to-left direction encounters and flows around the flat edges ofthe right triangular posts of the array (in this flow direction, theprofile of fluid flow through the gaps is symmetric about the axis ofthe gaps, being essentially parabolic).

B. Bump Arrays Having Gaps with Asymmetrical Flow Profiles

Described herein are bump array devices that are useful for segregatingparticles by size. In one embodiment, a device includes a body defininga microfluidic flow channel for containing fluid flow. An array ofobstacles is disposed within the flow channel, such that fluid flowingthrough the channel flows around the obstacles. The obstacles extendacross the flow channel, generally being either fixed to, integral with,or abutting the surface of the flow channel at each end of the obstacle.

The obstacles can be arranged in rows and columns, in such aconfiguration that the rows define an array direction that differs fromthe direction of fluid flow in the flow channel by a tilt angle (ε) thathas a magnitude greater than zero. The maximum operable value of ε canbe ⅓ radian. The value of c can be preferably ⅕ radian or less, and avalue of 1/10 radian has been found to be suitable in variousembodiments of the arrays described herein. The obstacles that are incolumns define gaps between themselves, and fluid flowing through theflow channel is able to pass between these gaps, in a direction that isgenerally transverse with respect to the columns (i.e., generallyperpendicular to the long axis of the obstacles in the column andgenerally perpendicular to a plane extending through the obstacles inthe column).

The obstacles can have shapes so that the surfaces (upstream of,downstream of, or bridging the gap, relative to the direction of bulkfluid flow) of two obstacles defining a gap are asymmetrically orientedabout the plane that extends through the center of the gap and that isparallel to the direction of bulk fluid flow through the channel. Thatis, the portions of the two obstacles can cause asymmetric fluid flowthrough the gap. The result can be that the velocity profile of fluidflow through the gap is asymmetrically oriented about the plane. As aresult of this, the critical particle size for particles passing throughthe gap adjacent to one of the obstacles can be different than thecritical particle size for particles passing through the gap adjacent tothe other of the obstacles.

A device can be made from any of the materials from which micro- andnano-scale fluid handling devices are typically fabricated, includingsilicon, glasses, plastics, and hybrid materials. The flow channel canbe constructed using two or more pieces which, when assembled, form aclosed cavity (preferably one having orifices for adding or withdrawingfluids) having the obstacles disposed within it. The obstacles can befabricated on one or more pieces that are assembled to form the flowchannel, or they can be fabricated in the form of an insert that issandwiched between two or more pieces that define the boundaries of theflow channel. Materials and methods for fabricating such devices areknown in the art.

In some cases, the flow channel can be preferably formed between twoparallel, substantially planar surfaces, with the obstacles formed inone of the two surfaces (e.g., by etching the surface to remove materialthat originally surrounded the non-etched portions that remain asobstacles). The obstacles can have a substantially constantcross-section along their length, it being recognized that techniquesused to fabricate the obstacles can limit the uniformity of the crosssection.

The obstacles can be solid bodies that extend across the flow channel,in some cases from one face of the flow channel to an opposite face ofthe flow channel. Where an obstacle is integral with (or an extensionof) one of the faces of the flow channel at one end of the obstacle, theother end of the obstacle can be sealed to or pressed against theopposite face of the flow channel A small space (preferably too small toaccommodate any of particles of interest for an intended use) can betolerable between one end of an obstacle and a face of the flow channel,provided the space does not adversely affect the structural stability ofthe obstacle or the relevant flow properties of the device. In someembodiments described herein, obstacles are defined by a cross-sectionalshape (e.g., round or triangular). Methods of imparting a shape to anobstacle formed from a monolithic material are well known (e.g.,photolithography and various micromachining techniques) andsubstantially any such techniques may be used to fabricate the obstaclesdescribed herein. The sizes of the gaps, obstacles, and other featuresof the arrays described herein depend on the identity and size of theparticles to be handled and separated in the device, as describedelsewhere herein. Typical dimensions are on the order of micrometers orhundreds of nanometers, but larger and smaller dimensions are possible,subject to the limitations of fabrication techniques.

As described herein, certain advantages can be realized by formingobstacles having sharp (i.e., non-rounded) edges, especially at thenarrowest part of a gap between two obstacles. In order to takeadvantage of the benefits of sharp edges, a skilled artisan willrecognize that certain microfabrication techniques can be preferable toothers for forming such edges. Sharpness of edges can be described inany of a number of ways. By way of example, the radius of curvature ofan edge (e.g., the vertex of a triangular post) can be measured orestimated and that radius can be compared with a characteristicdimension of the obstacle (e.g., the shorter side adjacent the vertex ofa triangular, square, or rectangular post, or the radius of a round posthaving a pointed section). Sharpness can be described, for example, as aratio of the radius of curvature to the characteristic dimension. Usingequilateral triangular posts as an example, suitable ratios includethose not greater than 0.25, and preferably not greater than 0.2.

In some cases, the number of obstacles that occur in an array is notcritical, but the obstacles can be sufficiently numerous that theparticle-separating properties of the arrays that are described hereincan be realized. In some cases, the precise layout and shape of thearray is not critical. In view of the disclosures described herein, askilled artisan in this field is able to design the layout and number ofobstacles necessary to make bump arrays capable of separating particles,taking into account the sizes and identities of particles to beseparated, the volume of fluid in which the particles to be separatedare contained, the strength and rigidity of the materials used tofabricate the array, the pressure capacity of fluid handling devices tobe used with the array, and other ordinary design features.

The obstacles can generally be organized into rows and columns (use ofthe terms rows and columns does not mean or imply that the rows andcolumns are perpendicular to one another). Obstacles that are generallyaligned in a direction transverse to fluid flow in the flow channel canbe referred to as obstacles in a column. Obstacles adjacent to oneanother in a column can define a gap through which fluid flows.Obstacles in adjacent columns can be offset from one another by a degreecharacterized by a tilt angle, designated ε (epsilon). Thus, for severalcolumns adjacent to one another (i.e., several columns of obstacles thatare passed consecutively by fluid flow in a single direction generallytransverse to the columns), corresponding obstacles in the columns canbe offset from one another such that the corresponding obstacles form arow of obstacles that extends at the angle ε relative to the directionof fluid flow past the columns. The tilt angle can be selected and thecolumns can be spaced apart from each other such that 1/ε (when ε isexpressed in radians) is an integer, and the columns of obstacles repeatperiodically. The obstacles in a single column can also be offset fromone another by the same or a different tilt angle. By way of example,the rows and columns can be arranged at an angle of 90 degrees withrespect to one another, with both the rows and the columns tilted,relative to the direction of bulk fluid flow through the flow channel,at the same angle of ε.

One or more portions of two obstacles that define a gap can be shaped insuch a way that the portions of the obstacles that are upstream from,downstream from, or bridging (or some combination of these, withreference to the direction of bulk fluid flow through the flow channel)the narrowest portion of the gap between the obstacles are asymmetricalabout the plane that bisects the gap and is parallel to the direction ofbulk fluid flow. Both for simplicity of fabrication and to aid modelingof array behavior, all obstacles in an array can be identical in sizeand shape, although this need not be the case. In some cases, allobstacles in an array are not identical in shape. Furthermore, arrayshaving portions in which obstacles are identical to one another within asingle portion, but different from obstacles in other portions can bemade.

Asymmetry in one or more portions of one or both of the obstaclesdefining a gap can lead to increased fluid flow on one side or the otherof the gap. A particle can be bumped upon passage through a gap only ifthe particle exceeds the critical particle size corresponding to thegap. The critical particle size can be determined by the density offluid flux near the boundaries of the gap (i.e., the edges of theobstacles that define the gap). Increased fluid flow on one side of agap (i.e., against one of the two obstacles defining the narrowestportion of the gap) can intensify flux density near that side, reducingthe size of the particle that will be bumped upon passage through thatside of the gap.

In one embodiment of the device, the shape of each of multiple obstaclesin a column can be substantially identical and symmetrical about theplane that bisects each of the multiple obstacles. That is, for any onecolumn of obstacles, the geometry encountered by particles traveling influid flowing through the gaps between the obstacles in the column canbe identical when the fluid is traveling in a first direction and whenthe fluid is travelling in a second direction that is separated from thefirst direction by 180 degrees (i.e., flow in the opposite direction).

The geometry encountered by particles traveling in fluid flowing throughthe gaps between the obstacles in the column can be different when thefluid is traveling in a first direction from the geometry encounteredwhen the fluid is travelling in a second direction that is differentfrom the first direction by 90-180 degrees. In this embodiment, fluidflow can, for example, be oscillated between the two flow directions,and the particles in the fluid can encounter the different obstaclegeometry. If these geometrical differences can result in different fluidprofiles through the gaps (compare the panels in FIG. 6B, for example),then the gap can exhibit different critical particle sizes in the twodirections. If a gap exhibits different critical sizes for flow in thetwo directions, then the populations of particles that will be bumpedupon passing through the gap can differ depending on the direction offlow. This difference in the populations bumped in the two directionscan be used to effect segregation of the differently-acting particles.

For example, consider a gap that exhibits a first critical size for bulkfluid flow in one direction, but exhibits a different critical size forbulk fluid flow in a second direction. For fluid flow in the firstdirection, particles having a size greater than the first critical sizecan be bumped, and particles having a size less than the first criticalsize cannot be bumped. Similarly, for fluid flow in the seconddirection, particles having a size greater than the second critical sizecan be bumped, and particles having a size less than the second criticalsize cannot be bumped. If flow is oscillated between the first andsecond directions, then particles having a size larger than both thefirst and the second critical sizes can be bumped in both directions.Similarly, particles having a size smaller than both the first and thesecond critical sizes cannot be bumped in either direction. For thesetwo populations of particles, flow oscillations of approximately equalquantities in both directions can leave these particles substantially attheir initial position. However, particles having a size intermediatebetween the two critical sizes can be bumped when bulk fluid flow is inone direction, but cannot be bumped when bulk fluid flow is in the otherdirection. Thus, when flow oscillations of approximately equalquantities in both directions are performed, these particles cannot beleft in their initial position, but can instead have been displaced fromthat original position by an amount equal to (the size of anobstacle+the gap distance G)×(the number of oscillations). In this way,these particles (the ones having a size intermediate between the twocritical sizes) can be segregated from the other particles with whichthey were initially intermixed.

When the first and second directions differ by 180 degrees (i.e., theflows are in opposite directions), the particles having a sizeintermediate between the two critical sizes can be displaced at an angleof 90 degrees relative to the direction of oscillating flow.

The behavior of particles in a bump array is not a function of theabsolute direction in which the particles (or the fluid in which theyare suspended) move, but rather can be a function of the array geometrythat the particles encounter. As an alternative to operating a bumparray with alternating flow between first and second directions, thesame particle-displacing effects can be obtained using flow only in thefirst direction by increasing the size of the array by two times thenumber of oscillations, maintaining one portion of the array in itsoriginal arrangement, but altering the second portion of the array suchthat the geometry of the array is identical to the geometry encounteredby particles in fluid moving in the second direction in the originalarray (even though the fluid moves in the first direction only). Usingthe array illustrated in FIG. 1 by way of example, the same displacementeffects on particles can be obtained by two oscillations of flow in thisarray (i.e., two units of flow left-to-right and two units of flowright-to-left) as can be obtained by four units of left-to-right flowthrough an array having four times the (left-to-right) length of thearray in FIG. 1, so long as two lengths of the array are arranged asshown in FIG. 1 and two lengths of the array are arranged as the mirrorimage (left-to-right) of the array shown in FIG. 1.

Described herein in a microfluidic device designed to separate objectson the basis of physical size. The objects can be cells, biomolecules,inorganic beads, or other objects of round or other shape. Typical sizesfractionated can range from 100 nanometers to 50 micrometers; smaller orlarger sizes can be fractionated. Use of these arrays can involvecontinuous flows in one direction, and particles can be separated fromthe flow direction by an angle which is a monotonic function of theirsize.

By changing the shape of the posts from circles to a shape that isasymmetric about an axis parallel to the fluid flow, functionalities maybe added:

1. The critical particle size for bumping may be different depending onwhich direction a particle moves through the array. This has beenexperimentally verified with right triangular posts, and extends toarbitrary shapes that are asymmetric about the flow axis.

2. With such designs, the angle of displacement from the fluid flow ofparticles may be designed not to be monotonic—e.g. peaked at a certainparticle size.

Such bump arrays have multiple uses, including all of the uses for whichbump arrays were previously known.

The device can be used to separate particles in a selected size band outof a mixture by deterministic lateral displacement. The mechanism forseparation can be the same as the bump array, but it can work underoscillatory flow (AC conditions; i.e., bulk fluid flow alternatingbetween two directions) rather than continuous flow (DC conditions;i.e., bulk fluid flow in only a single direction). Under oscillatoryflow, particles of a given size range can be separated perpendicularlyfrom an input stream (perpendicular to the alternating flow axis whenthe alternating flows differ in direction by 180 degrees) without anynet displacement of the bulk fluid or net displacement of particlesoutside the desired range. Thus, by injecting a sample includingparticles of the given range into an obstacle array and thereafteralternating fluid flow through the obstacle array in opposite directions(i.e., in directions separated from one another by 180 degrees),particles that exceed the critical size in one flow direction but do notexceed the critical size in the other flow direction can be separatedfrom other particles in the sample by the bumping induced by the array.Such particles can be bumped (and follow the array direction) when fluidflows in one direction, but are not bumped (and follow the bulk fluidflow direction) when fluid flows in the opposite direction. Particlesthat do not exceed the critical size in either flow direction will notbe bumped by the array (i.e., will follow the bulk fluid in bothdirections), and will remain with the sample bolus. Particles thatexceed the critical size in both flow directions will be bumped by thearray (i.e., will follow the array direction) when fluid flows in onedirection, and are also bumped (i.e., will follow the array direction inthe opposite direction) when fluid flows in the opposite direction, andwill therefore remain with the sample bolus.

Critical particle size can depend on direction of fluid flow.Intermediate sized particles can be made to ratchet up a device underoscillatory flow.

Second, in a continuous flow mode, particles of a desired size can beinduced to move to one side of a fluid stream, and particles above orbelow that size to the other side or not displaced at all. Thuscollection of desired particles can be easier. In conventional devices,particles above a desired range are also displaced from the fluid flowto the same side of the flow, so separating the desired from undesiredlarger ones can be harder. In this embodiment, obstacles definingdifferent critical sizes for fluid flow in opposite directions areemployed in two configurations that are mirror images of one another.For example, with reference to FIG. 1, such an array would include righttriangular posts arranged as shown in FIG. 1 (i.e., hypotenuse slopingfrom lower right to upper left and the tilt angle ε extending from thehorizontal toward the bottom of the figure) and would also include righttriangular posts arranged as they would appear in a mirror heldperpendicularly at the right or left side of the array shown in FIG. 1(i.e., right triangular posts having their hypotenuse sloping from upperright to lower left and the tilt angle ε extending from the horizontaltoward the top of the figure). Particle separation achieved by bulkfluid flow in a single direction (i.e., either from left-to-right orright-to-left) through such an array would be equivalent toback-and-forth flow through the array illustrated in FIG. 1. Particlesin the selected size range can be bumped toward the top of the array (asshown in FIG. 1), while particles having larger or smaller sizes canremain at the vertical level at which they enter the array (assumingapproximately equal numbers of obstacles in each of the twoconfigurations are encountered).

Reduction in critical particle size as a ratio of gap, compared tocircular posts, can occur when particles bump off sharp edges. This canallow larger separation angle without fear of clogging the device fasterseparations.

These developments can reduce the necessary chip area compared to acontinuous flow bump array.

A device described herein can be a microfabricated post arrayconstructed using standard photolithography. A single mask layer can beetched into silicon or used to make a template for PDMS molding.Usually, post arrays can be sealed with a PDMS coated cover slip toprovide closed channels

Oscillatory flow operation can require more complicated fluid controldrivers and interfaces than continuous flow operation.

FIG. 11 is a scanning electron microscope image of posts in an obstaclearray of a type described herein. Right isosceles triangular posts, 6microns on a side, were placed on a square lattice with spacing of 10microns, giving a gap of approximately 4 microns. The square lattice wastilted 5.71 degrees (0.1 radians) with respect to the device sidewallsto provide necessary asymmetry. Fluid flows along the horizontal axis.

In FIG. 1, the total fluid flux through each gap can be divided inton=1/ε′ flow streams (stream tubes), where n is a whole number. Each flowstream can carry equal fluid flux, shown schematically in FIG. 1 forn=3. The stream tubes can be separated by stall lines, each stall linebeginning and ending on a post. The stream tubes shift their positionscyclically so that after n rows each streamline returns to its initialposition within the gap.

The width of the stream closest a post can determine the criticalparticle size. If the particle's radius is smaller than the width of thestream, then the particle's trajectory can be undisturbed by the postsand travel in the same direction of the flow. If the particle's radiusis larger than the width of the closest stream, then it can be displacedacross the stall line and it's trajectory can follow the tilted axis ofthe array (i.e., the array direction).

The width of the stream closest to the post can be determined byassuming that the velocity profile through a gap is parabolic—the casefor fully-developed flow in a rectangular channel. Since each stream cancarry equal flux and there are n streams, integration can be done overthe flow profile such that the flux through a stream of width Dc/2 (Dcis the critical diameter of a particle) closest to the post is equal tothe total flux through the gap divided by n. That is, the integral from0 to Dc/2 of u(x) dx (u being a function of flux at any position xwithin the gap) being equal to 1/n times the integral of u(x) dx overthe entire gap.

Thus, the critical particle size can be determined from the flowprofile. For the case of circular posts, a parabolic flow profile canclosely approximates the flow profile through the gap and the criticalparticle size can be determined analytically.

FIG. 4A shows a numerical simulation of flow profile for an array oftriangular posts. In some cases, it cannot be assumed that the flowprofile through triangular posts is parabolic because of the brokensymmetry. Therefore, flow profile through gap of triangular posts wasextracted from numerical simulation (program—COMSOL) of flow through anarray with same size and spacing as devices actually made.

FIG. 4B illustrates a comparison of velocity flow profiles betweencircular and triangular posts. Normalized velocity profiles through gapbetween triangular and circular posts are shown. As shown, the flowprofile for the triangle posts is asymmetric about the center of thegap; more fluid flows along the vertex of the triangle than along theflat edge.

FIGS. 12-14 illustrate particle motion in a ratchet bump array of a typedescribed herein. When particles move through the array, the side of thepost they interact with depends on which direction they are moving inthe array. In this case, when the particles are moving fromright-to-left, they bump off the flat edge of the triangular posts. Whenthe particles are moving from left-to-right, they bump off the sharpvertex of the triangular posts. Thus, since the flow profile isasymmetric, it cannot be expected that particles follow the sametrajectory when travelling in both directions through the array.

Critical Particle Size for Triangular Posts—Employing the same kind ofanalysis described in the Inglis et al., 2006, Lab Chip 6:655-658,integratation can occur over the flow profile to find the width ofcharacteristic streams. However, since the flow profile is asymmetricabout the center of the gap, the stream width, and hence the criticalparticle size can be different depending on which side is examined. Asshown in FIG. 4B, the result of the asymmetry introduced by thetriangular posts is that the critical particle size can be differentdepending on which side of the triangular obstacle particles interactwith. If they are moving along the sharp vertex, then the criticalparticle size can be smaller than if they are moving along the flatedge. Critical particle size versus array angle (ε) are plotted in FIG.15 compared to circular posts. The critical particle size for bumpingalong the sharp vertex of the triangle can be substantially smaller thanfor that of circular posts or the flat edge. This can allow higherangles of separation to be used without fear of clogging the devices.When the particle diameter is larger than the gap size (G in FIG. 1),there can be substantial risk that the array will become clogged ifparticle density is high.

FIGS. 3A-3C illustrate representative particle behavior in a ratchetbump array. For a device constructed as shown in FIG. 11, threerepresentative particles were chosen for this illustration. One particle(illustrated in FIG. 3B) was chosen larger than both critical particlesizes (i.e., larger than the critical particle sizes defined byright-to-left and left-to right fluid flows). One particle (illustratedin FIG. 3A) was chosen that was smaller than both critical particlesizes. Finally, one particle (illustrated in FIG. 3C) was chosen in theintermediate range i.e., smaller than the critical particle size (D_(F)in FIG. 12) along the flat edge, but larger than the critical particlesize (D_(V) in FIG. 12) along the sharp edge. These figures illustratethe behavior of particles that were put into the device and theirtrajectory under oscillatory flow was observed.

Large Particle (FIG. 3B): Since the particle is larger than the criticalparticle size along both edges, it follows the array tilt axis (E) inboth directions and shows no net displacement under oscillatory flow.

Small Particle (FIG. 3A): Since the particle is smaller than thecritical particle size along both edges, it follows the fluid trajectoryin both directions and shows no net displacement.

Intermediate Particle (FIG. 3C): When the particle moves to the right,it bumps off the flat edge of the triangular posts. Since it is smallerthan the critical particle size (D_(F)), it follows the fluidtrajectory. When the particle moves to the left, it bumps off the sharpvertex of the triangular posts. Since it is larger than the criticalparticle size on this side (D_(V)), it follows the array tilt axis andis displaced upward. As shown, under oscillatory flow, particles in theintermediate range are displaced perpendicular to the direction of theflow. After three cycles of moving back and forth, the bulk fluid hasnot been displaced, but the particle has moved over 200 microns.

If all three particle types were mixed and placed in a single arrayunder oscillatory flow (i.e., fluid flow oscillating between theright-to-left and left-to-right directions), the intermediate particleswould be displaced toward the top of these figures while the small andlarge particles would have no net motion.

In FIGS. 12-14, representations of intermediate, small, and largeparticles (respectively) were overlaid on top of numerical simulation ofstream tubes to show motion of particles more clearly. n=1/ε was chosento be 3 to allow periodicity to be seen more easily.

When intermediate particles (FIG. 12) travel along the sharp edge, theybump as can be expected. However, when the particles travel along theflat edge, their motion can be different from that of the smallparticles. When they perform their characteristic zig to continue in thesame direction as the fluid, they are too large to stay in that streamthat is close to the sharp vertex and they are displaced across thefirst stall line. The result is that their motion is periodic in tworows instead of three. With any other tilt angle, the motion can besimilar and the periodicity is n−1. The result of this n−1 periodicityis that the intermediate sized particles are actually displaced againstthe axis tilt angle. Thus a mixture of large, small and intermediateparticles will be separated into three streams. Small particles will gostraight through (see FIG. 13). Large particles will follow the arraytilt axis (see FIG. 14). Intermediate particles will follow a separatepath that is dependent on the post geometry.

The applications for which devices described herein are useful includethe same ones described in the Huang patent (U.S. Pat. No. 7,150,812):biotechnology and other microfluidic operations involving particleseparation.

Continuous-flow fractionation of small particles in a liquid based ontheir size in a micropost “bump array” by deterministic lateraldisplacement was demonstrated previously (e.g., Huang et al., 2004,Science 304:987-990). A ratchet bump array described herein can possessall the same advantages of the previous work, but can add two newfunctionalities:

First, the devices can be used to separate particles in a selected sizeband out of a mixture by deterministic lateral displacement underoscillatory flow (AC conditions) rather than continuous flow (DCconditions). Under oscillatory flow, particles of a given size range canbe separated perpendicularly from an input stream (perpendicular to theAC flow axis) without any net displacement of the bulk fluid orparticles outside the desired range.

Second, in continuous flow mode, the device can exhibit trimodalbehavior. Particles of a desired size range can be induced to move toone side of a fluid stream, and particles above or below that size tothe other side or not displaced at all. Thus collection of these desiredparticles may be easier. In conventional devices, the devices werebimodal and all particles above a desired size range are displaced fromthe fluid flow to the same side of the flow, so separating the desiredfrom undesired larger ones can require multiple stages whereas theratchet bump array can require only one.

As used herein, each of the following terms can have the meaningassociated with it in this section.

The terms “bump array” and “obstacle array” are used synonymously hereinand can describe an ordered array of obstacles that are disposed in aflow channel through which a particle-bearing fluid can be passed.

A “substantially planar” surface can be a surface that has been madeabout as flat as a surface can be made in view of the fabricationtechniques used to obtain a flat surface. In some cases, no fabricationtechnique will yield a perfectly flat surface. So long as non-flatportions of a surface do not significantly alter the behavior of fluidsand particles moving at or near the surface, the surface can beconsidered substantially planar.

In a bump array device, “fluid flow” and “bulk fluid flow” can be usedsynonymously to refer to the macroscopic movement of fluid in a generaldirection across an obstacle array. These terms do not take into accountthe temporary displacements of fluid streams for fluid to move around anobstacle in order for the fluid to continue to move in the generaldirection. In some cases, the bulk fluid flow across an obstacle arrayas provided herein comprises two or more fluid streams flowing from aninput or inlet portion or area of the array to an output or outletportion or area of the array. Each of the two or more fluid streams canflow parallel to each other. The two or more fluid streams can becomprised of the same or different components. The components can be anybuffer or reagent described herein or known in the art. In some cases,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20fluid streams flow in parallel from an input or inlet portion or area ofan array as provided herein to an output or outlet portion or area ofthe array. Fluid stream can also be referred to as a “stream tube”.

In a bump array device, the tilt angle ε can be the angle between thedirection of bulk fluid flow and the direction defined by alignment ofrows of sequential (in the direction of bulk fluid flow) obstacles inthe array. This angle is illustrated in FIGS. 1, 6, and 11, for example.

In a bump array device, the “array direction” can be a direction definedby the alignment of rows of sequential (in the direction of bulk fluidflow) obstacles in the array.

A “critical size” or “predetermined size” of particles passing throughan obstacle array can be a parameter that describes the size limit ofparticles that are able to follow the laminar flow of fluid nearest oneside of a gap through which the particles are travelling when flow ofthat fluid diverges from the majority of fluid flow through the gap.Particles larger than the critical size can be ‘bumped’ from the flowpath of the fluid nearest that side of the gap into the flow path of themajority of the fluid flowing through the gap. In a bump array device,such a particle can be displaced by the distance of (the size of oneobstacle+the size of the gap between obstacles) upon passing through thegap and encountering the downstream column of obstacles, while particleshaving sizes lower than the critical size (or predetermined size) willnot necessarily be so displaced. When a profile of fluid flow through agap is symmetrical about the plane that bisects the gap in the directionof bulk fluid flow, the critical size can be identical for both sides ofthe gap; however when the profile is asymmetrical, the critical sizes ofthe two sides of the gap can differ. When assessing a non-sphericalparticle, its size can be considered to be the spherical exclusionvolume swept out by rotation of the particle about a center of gravityin a fluid, at least for particles moving rapidly in solution. The sizecharacteristics of non-spherical particles can be determined empiricallyusing a variety of known methods, and such determinations can be used inselecting or designing appropriate obstacle arrays for use as describedherein. Calculation, measurement, and estimation of exclusion volumesfor particles of all sorts are well known.

A particle can be “bumped” in a bump array if, upon passing through agap and encountering a downstream obstacle, the particle's overalltrajectory follows the array direction of the bump array (i.e., travelsat the tilt angle ε relative to bulk fluid flow). A particle is notbumped if its overall trajectory follows the direction of bulk fluidflow under those circumstances. Conceptually, if flow through a gap isvisualized as being composed of multiple individual layers of fluid(i.e., stream tubes, if thought of in a cross-section of fluid flowingthrough the gap), a particle can be “bumped” if the particle isdisplaced by a post out of its incident flow tube into an adjacent flowtube as it traverses a gap bounded by the post.

“The direction of bulk fluid flow” in an obstacle array device can referto the average (e.g., macroscopic) direction of fluid flow through thedevice (i.e., ignoring local flow deviations necessitated by flow aroundobstacles in the fluid channel)

C. A Deterministic Microfluidic Ratchet

This example describes a microfluidic device in which the trajectory ofparticles within a certain size range varies with the direction theparticles move through the device. This ratcheting effect can beproduced by employing triangular rather than the conventional circularposts or microposts or obstacles in a deterministic lateral displacement(DLD) device where an array of posts selectively displaces particles asthey move through the array. This effect can then be used to demonstratea fractionation technique where particles can be separated from a fluidplug without any net motion of the original fluid plug. The underlyingmechanism of this method can be based on an asymmetric fluid velocitydistribution through the gap between posts.

Microfluidic devices, such as those used in “lab on a chip”applications, can operate at low Reynolds number (“low” Reynolds numberrefers to Reynolds number not greater than 1, and preferably smaller,such as 0.1, 10⁻³, or smaller). In this regime, the fluid flow throughan arbitrary geometry can be considered to be time-invariant reversingthe applied pressure gradient that drives the fluid will reverse theflow field because inertial effects are negligible. At high Pecletnumber (“high” Peclet number can refer to Peclet number greater than 1,and preferably much greater, such as 10, 100, or more), this can beextended to say that diffusive effects can be ignored and objects in thefluid will deterministically flow along a stream tube unless some otherinteraction, such as displacement by steric repulsion from a channelwall, disrupts their path and moves them to an adjacent stream tube. Thedegree to which the particle trajectory can be shifted from its originalpath can depend directly on its size; larger particles can be displacedfarther than smaller particles and can consequently follow differentstream tubes as they progress through the device. This phenomenon, whichcan be called deterministic lateral displacement (DLD), has been used inseveral schemes to perform microscale particle separations.

A “bump array” can be a microfluidic device that relies on deterministiclateral displacement (DLD) to separate particles with high resolution.This device can rely on asymmetric bifurcation of fluid streams in apost array that is tilted at an angle ε (epsilon; typically on the orderof 0.1 radians) with respect to the direction of the overall fluid flow.The fluid flowing through a gap splits around a post in the next row,with 1/ε of the fluid going through the gap on one side of the nextpost, while the other ε of fluid goes around the other side of the nextpost. As a result, the fluid motion can be characterized by 1/ε streamsthat cycle through positions in the gap, but travel straight on average.If a particle suspended in the fluid is small compared to the width of astream in a gap, the posts will not affect it as it moves through thearray and it can travel straight with the fluid flow. However, if theparticle is large relative to the width of a stream, it can be displacedinto an adjacent stream when the stream it occupies is nearest a post asit moves through a gap. Because of the cyclical way the stream can movethrough gaps, this displacement or “bump” can occur at every row and theparticle can travel at an angle with respect to the fluid and othersmall particles. With a sufficiently long device, significant separationcan be obtained between large and small particles.

FIG. 2A shows a fluorescent time-lapse image of a small particle (1.1micron diameter polystyrene bead) flowing through such an array at atypical speed of 100 microns/sec. As the particle moves forward, ittakes many small steps parallel to the array axis as it moves through,followed by one larger step perpendicular to the motion of the fluid (inwhat we refer to as “zig-zag mode”), so that the overall motion is tofollow the plug of fluid which originally contained the particle. Intaking the image of FIG. 2A, the fluid flow was cycled back and forth(by reversing the pressure) twice. The particle retraced its path, asexpected from flows at low Reynolds and high Peclet number in adeterministic device not relying on diffusion.

FIG. 2B shows a similar image but for a larger particle (3.1 microns).In this case the particle clearly follows the array axis (i.e., travelsin the array direction) and not the fluid flow. Because the particle isdisplaced from its flow path by the posts in each row, this can bereferred to as “bumping mode.” This difference in flow direction as afunction of particle size can be exploited to make fractionation devicesfor polystyrene beads as well as biological particles. As in FIG. 2A,the time lapse image shows the path of the particle over two cycles offlowing forward and back, and again the path of the particles isreversible (i.e., the particles end up where they began).

FIG. 2C shows the same experiment in the same array for a particle ofintermediate size (1.9 microns). The results are very different fromthose shown if FIGS. 2A and 2B. This particle “zig-zags” when going tothe right (i.e., moving from left-to-right) to follow the fluid flow but“bumps” when going to the left to follow the post array axis. Its pathis not reversed when the fluid flow direction is reversed, with the netresult that such particles are separated from a plug of fluid in aperpendicular direction when the fluid is subjected to an oscillatoryflow.

The displacement of a particle off of a post can be an inherentlyirreversible interaction, but particle trajectories in a circular postbump array are ostensibly reversible because of symmetry. There is nocontroversy in this statement for small particles which follow the fluidbecause the fluid flow must be reversible in the low Reynolds numberregime (typical Re 10e-3 for fluid velocity 100 microns/sec and lengthscale 10 microns). However, large particles do not follow the fluid;instead, they are displaced off posts by steric repulsion so even thoughthe fluid can reverse direction, the trajectory of particles whichinteract with the posts will not necessarily be reversible unless theirinteraction with the posts is symmetric with the direction of the fluid.In the schematic in FIG. 3A, particles moving to the left are displaceddownward by the top row of posts while particles moving to the right aredisplaced the same amount by the bottom row of posts. However, if theimage is rotated 180 degrees, which is analogous to switching thedirection of the fluid, the situation is exactly switched, so the resultmust be the same in either direction. This rotation works because boththe lattice points and post shape are invariant under 180 degreerotation. As a result, both large and small particles in bump array withcircular posts can retrace their steps if the flow is switched back andforth.

Numerical simulations showed that the velocity profile through a gapbetween triangular posts was shifted towards the side of the gap withthe vertex. The fluid velocity profile through a gap between postsdepends strongly on the local geometry at the gap. For the case of thetriangular posts presented here, where there is a sharp vertex on thebottom and a flat edge on the top, a significant deviation from theparabolic flow profile used to describe pressure-driven flow throughcircular posts should be expected. FIG. 4A shows a numerical simulationof the fluid velocity in an array like that used to produce the particletrajectories in FIG. 2, along with a cross section of the velocityprofile across the gap. The line was placed across the smallest spacingbetween posts to correspond with the narrowest stream widths wherecrossing stall lines is most likely to occur. The vertices of thetriangle were rounded off with a curvature of 500 nm to approximate therounding off of a sharp point that results from optical lithography. Itwas found that the flow profile was invariant under changes in the arraytilt so this flow profile can be assumed to be the general flow profilefor triangular posts arranged in this way.

FIG. 4B shows a comparison between the flow profiles of triangular andcircular posts. For round posts, the profile is nearly parabolic asexpected for Poiseuille flow through an infinitely long one-dimensionalchannel. For triangular posts, however, the flow profile is biasedtowards the sharp triangular corner pointing up into the flow stream. Inother words, the streams bunch closer together near this vertex and thecritical particle size for a particle to be bumped across a stall lineis smaller than it would be for an array with the same gap size but withround obstacles. Along the flat edge, the opposite is true. Because thefluid travels preferentially along the vertex, the width of the streamalong the flat edge is wider than for circular posts. The effect of thetriangular posts is to create two separate critical particle sizes, onefor moving along the vertex of the triangle and another for moving alongthe flat edge. Therefore, particles in between these two criticalparticle sizes can exhibit different behavior depending on whichdirection they are moving through the array. To show this, a techniqueused by Inglis et al., 2006, Lab Chip 6:655-658 was employed to estimatethe critical particle size for circular posts by using the extractedvelocity profile instead of the parabola assumed for circular posts.

FIG. 5 shows this calculation of the critical particle size as a ratioof the gap for the vertex and flat of the triangle as well as forcircular posts versus array tilt angle. The particles shown in figuretwo are shown as circles on the plot. They show good agreement with thepredicted behavior. The 1.1 micron bead is smaller than both criticalparticle sizes so it travels with the fluid in both directions and showsno net displacement when the fluid direction is cycled. The 3.1 micronparticle is bigger than both critical particle sizes so it is displacedalong the array axis in both directions and shows no net displacementwhen the fluid direction is cycled. The 1.9 micron particle is inbetween the two critical particle sizes so it travels with the fluidwhen it moves along the flat edge of the triangle and with the arrayaxis when it moves along the vertex of the triangle. As a result, itshows a net displacement when the fluid flow is cycled. This ischaracteristic of a ratcheting behavior. With no net displacement of thefluid, particles in the intermediate range of an array show a netdisplacement after several fluid flow oscillations. This ratchet differsfrom other ratchets in that the ratcheting motion does not occur alongthe axis of the applied force corresponding to fluid flow in eitherdirection. Rather, it is perpendicular to the motion of the fluid.

D. Bump Array Employing Triangular Posts

This example describes microfluidic arrays which sort particles based onsize according to the deterministic lateral displacement (DLD) method,by using triangular posts instead of round or circular posts. Whentriangular posts are used rather than round posts, and the triangularposts are properly oriented (i.e., such that the surfaces defining thegap are asymmetric), the critical size is decreased for a given gap sizebetween the posts. This is because the different post geometry on eitherside of the gap causes an asymmetric flow profile through the gap, withflux shifting towards the vertex of the triangle. This shift in fluidflux reduces the width of the stream that determines the criticalparticle size. In this example, both experiment and modeling are used toshow that changing the post shape from circular to triangular results inseveral practical advantages over similar arrays with circular postsincluding increased dynamic range and throughput.

Deterministic lateral displacement can be a size-based particleseparation technique that relies on selective displacement of particlesby an array of obstacles disposed in a flowing fluid. FIG. 6Aillustrates a schematic of the relevant array parameters and importantfeatures of the devices described in this example. The obstacle array iscomposed of columns of posts in which each adjacent column is offset asmall distance with respect to larger channel walls that dictate thedirection of bulk fluid flow (“FLUID” in FIG. 6A). In this case, theposts are equilateral triangles with side length S (contrary to FIG. 6A,S is the side length, not the distance from a vertex of the triangle tothe base opposite that vertex). This offset produces an array where anaxis along which the obstacles are situated is situated at a tilt angleε with respect to the direction of fluid flow. The tilt angle isselected such that the array is periodic after 1/ε rows. In this case,the fluid flowing through gaps between posts (length of gap isdesignated G in FIG. 6A) can be partitioned into an integer number ofstream tubes delineated by stagnation streamlines. Constrained by theperiodicity and the direction of average fluid flow, each of thesestream tubes carries an equal volumetric flux.

Particles suspended in the fluid exhibit one of two behaviors dependingon their size relative to the width of stream tube nearest to the postas they move through a gap. Unperturbed by other effects, particles canroughly follow the stream tubes in the fluid flow. This behavior can beobserved for particles having radii narrower than the stream tube width.These particles, shown as the lower particle and dotted trajectory inFIG. 6A, are not affected by the posts and weave through the array whileremain within the bounds of a single stream. As a result, they travel onaverage in the same direction as the bulk fluid flow. Particles havingradii larger than the stream tube width, denoted as the upper particleand dotted trajectory in FIG. 6A, do not fit within a single stream tubeas they travel through the gap. Those larger particles aredeterministically displaced by the post across the stagnation streamlineinto the adjacent stream tube. Because of the way the stream tubes cyclethrough their position in the gap, this displacement will occur at everycolumn of posts and the larger particle will travel along the array axis(i.e., in the array direction, which differs from the bulk fluiddirection by the tilt angle _(E)). This binary behavior leads us todescribe a critical size which separates these two behaviors. As theparticles to be separated are most frequently described by theirdiameter, we denote the critical size as twice the width of the streamtube nearest to the post in the gap between posts.

Changing the post shape can have a strong effect on the criticalparticle size by changing the shape of the flow profile through the gap.Alterations to the flow profile alter the width of the stream tubesnearest the posts that define a gap. Because critical particle size canbe directly related to these widths, alteration to the flow profilewithin a gap also alters the critical size(s) defined by the gap. Bychanging the cross-sectional shape of the posts from a circular shape toequilateral triangles, an asymmetry can be created in the flow profilethrough the gap that shifts more fluid flux towards the triangle vertex,as shown in FIG. 6B. This results in different stream tube widths at thetop (adjacent the flat edge of a triangular post) and bottom (adjacentthe vertex of a triangular post) of the gap and gives the array twodistinct critical particle sizes.

The shift in flux towards the vertex of the triangle can lead to areduced stream tube width along this edge and hence can reduce thecritical particle size corresponding to that stream tube and edge,relative to a similar array with circular posts. This is demonstrated inthe two panels of FIG. 6B, which shows numerically simulated flowprofiles across the gaps. The two flow profiles, normalized to the widthof the gap between posts and the maximum velocity, are plotted side byside for comparison. The fluid constituting the first stream tube fortilt angle ε= 1/10 has been shaded to emphasize the difference in streamwidth, decreasing from about 20% of the gap bounded by circular posts toabout 15% of the gap bounded by triangular posts. This shift is centralto the reduction in critical particle size behavior exhibited by deviceswith triangular posts. The shifted flow profile created by triangularposts can be used to create a deterministic microfluidic ratchet. In theinformation discussed in this example, the focus is on improvement tocontinuous flow particle separation devices and the deterministiclateral displacement of particles within them that are enabled bychanging the post shape.

The reduction in critical particle size enabled by triangular posts wascharacterized by examining the behavior of fluorescent beads of inarrays with various amounts of array tilt and comparing the results totheoretically predictions. FIG. 7 shows observed particle behavior(displaced by the array or not displaced by the array) normalized to thegap size versus array tilt as well as predicted critical particle sizesusing the method described by Inglis et al., 2006, Lab Chip 6:655-658.The lines in FIG. 7 represent the predicted critical particle size for agiven tilt angle the solid line representing predictions for arrays withtriangular posts and the dotted line representing predictions for arrayswith round posts. Particles above the line are expected to be displacedby the array, particles below the line are not expected to be displaced.The data demonstrated that there is reasonable agreement with thepredicted behavior for higher tilt angles while there is some deviationat the shallower tilt angles, especially at a tilt angle ε of 1/20radians. This deviation could be caused by the flow through the arraynot being completely horizontal, which will have a large affect atshallower array tilts, or because of rounding of the triangular postedges, which will be discussed later in this example.

The predicted particle behavior for circular posts, signified by thedotted line, has been added as a comparison. For any practical tiltangle (between ⅕ and 1/100), the critical size in an array withtriangular posts can be substantially smaller than the critical size ina similar array with circular posts, the difference amounting to up to10% of the gap for the steeper tilt angles. These properties allowsmaller particles to be separated by an array of triangular posts thancan be separated by an array of round posts having the same gap spacing.These properties also mean that the gap spacing for triangular poststhat is necessary to separate particles of a selected size is largerthan the corresponding gap spacing for round posts that would benecessary to separate the same particles.

In either case, a reduced critical particle size as a fraction of thegap can be useful in reducing clogging in the array. In some cases,biological samples contain species with a broad range of sizes. In somecases, filtering or multiple separation stages can be used to ensurethat an array continues to function. Using triangular posts allows oneto increase the size of the gap for a given critical particle size andreduce the chances that the array will clog. FIG. 8 illustrates how muchlarger the gap between posts can be made as a function of the arraytilt. Plotted as a ratio of the two gaps for a fixed critical particlesize, a minimum 20% improvement can be seen with increasing gap size asthe tilt is reduced, with a ratio of 1.25 for a tilt angle of ¼ and aratio of 1.94 for a tilt angle of 1/100. Thus, shallower tilt angles canfacilitate use of larger gaps at the cost of a smaller separation angleand increased array size. However, larger gaps can provide anotherbenefit in terms of increased array throughput.

A throughput comparison between an array with triangular and circularposts showed a substantial increase in average velocity for a givenpressure drop in the array with triangular posts. Arrays with triangularposts or with circular posts were constructed with nearly identicalcharacteristics. They each had the same overall channel width andlength, depth, tilt angle ( 1/10), and post size (the diameters of roundposts were equal to the side lengths of the equilateral triangularposts). The single variation was the gap between posts, which wasdesigned and verified with numerical simulation to give a criticalparticle diameter of approximately 3.2 microns for both arrays. Thosenumerical simulations indicated that the critical particle diameter wasachieved using a gap of 10.5 microns in arrays with triangular posts anda gap of 8.3 microns in arrays with circular posts.

The trajectories of 500 nanometer fluorescent beads were recorded withan electron multiplying charged coupled device (EMCCD) camera capturingvideo at 10 frames per second and then analyzed using MATLAB™ softwarefor a given pressure gradient across the array.

Small particles that would not be displaced (i.e., bumped) by the arraywere chosen so they would sample each of the flow streams evenly andprovide an accurate representation of the overall average fluidvelocity.

The average particle velocities are plotted in FIG. 9 as a function ofpressure gradient along with a weighted linear fit. The fitted linesdemonstrate that particles in the triangular post array moved muchfaster. The upper range of pressures was limited by the field of view ofthe microscope and the capture speed of the camera. Beyond several kPain pressure, the particles traversed the entire field of view within oneor two frames of the video and no accurate estimate of velocity could bemade. However, since the Reynolds number in these experiments is on theorder of 10⁻², the linear fit can safely be extended into the tens ofkPa range to match the expected linear relationship between velocity andpressure that is seen for low Reynolds number flows. The posts need notbe triangular in cross-section. Posts having other (square, oblong, orirregular) cross-sectional profiles can also be used, so long as theshape of the obstacles causes the gap to be asymmetric.

Comparing the slopes of the two linear fits in FIG. 9, it can be seenthat particles in the array with triangular posts traveled 85% faster onaverage than those in an array with circular posts. This result agreeswith numerical simulation performed with COMSOL™ software that showedthat the average velocity for was 82% faster for triangular posts. Themechanism behind these findings can be understood by drawing an analogyto Poiseuille flow between two parallel plates, where the averagevelocity for a fixed pressure gradient is proportional to the smallestdistance between the plates squared. The analogy is not exact becausethe confining structure is an array of posts instead of two parallelplates, but underscores the benefits of increasing the width of the gap,where just a few microns yields a substantial increase in throughput.

The gains achieved by changing the post shape are degraded if care isnot taken to maintain sharp post vertices. FIG. 10 shows the effect ofrounding triangular post edges on the critical particle size. An arraywith 10 micron posts, 10 micron gaps between posts, and tilt angle of ⅓owas simulated using COMSOL™ software, with the vertices rounded tovarious radii of curvature ranging from none (r=0) to complete roundingwhere the final shape is a circle (r=S/12^(1/2)). Flow profiles acrossthe gaps were extracted for each rounding and the critical size for thegiven tilt was calculated using previously stated methods. As shown inFIG. 10, there is a dramatic increase in the critical particle size asthe post shape transitions from triangular to circular. Starting at0.174 G when the post is completely triangular (i.e., r=0), criticalparticle size increases 35% to 0.235 G when the post is completelycircular (r=S/12^(1/2)). The transition suggests that if a fabricationprocess that produces an undesirable vertex rounding, using larger posts(increasing S) will help to maintain the decreased critical particlesize that results from using triangular posts.

This observation also helps to explain the deviation from expectedbehavior observed for some of the fluorescent beads in FIG. 7. SEMimages of the posts show vertex rounding (r/S) of 0.118±0.006, whichcorresponds to an increase in the critical particle size from 0.93microns to 1.12 microns.

E. “Car Wash” Device

In one aspect, the devices, methods, compositions, and kits providedherein replace chemical or reagent treatment and/or manualwash/concentrate steps present in many techniques known in the art. Thedevices provided herein can replace any procedures requiringcentrifugation known in the art. In some cases, the devices, methods,compositions, and kits provided herein replace the labeling (e.g., cellsurface and/or intracellular), and wash/concentrate steps used toprocess samples comprising cells. The processed cells can then be usedfor research and/or clinical diagnostic testing. In some cases, thedevices, methods, compositions, and kits provided herein replace thelabeling (e.g., cell surface and/or intracellular), wash/concentrate,and/orerythrocyte lysissteps used to process blood samples for use inresearch and clinical diagnostic testing. The clinical and diagnostictesting can be used for cancer, infectious and inflammatory diseasesand/or many other diseases. In some cases, the devices, methods,compositions, and kits provided herein replace the lysis, enzymatictreatment(s), cloning, and/or wash/concentrate steps used to processsamples comprising cells from cells to nucleic acid libraries. Thenucleic acid libraries can be used in sequencing. The sequencing can beany Next Generation sequencing method or platform known in the art. Themethods provided herein can be automated reagent-free microfluidicprocesses that can effectively harvest, wash and concentrate particles(e.g., cells) in several minutes, with high yield, high reproducibility,and low cost. The particles can be any of the cells provided herein. Insome case, the particles are stem cells, leukocytes and/or leukemiacells.

As shown in FIG. 17B, a “car wash” device as provided herein cancomprise two inlets (FIG. 17B; ‘sample’ and ‘buffer’) which areconfigured to flow two separate fluids from an input area of a device toan output area of the device. In FIG. 17B, the two fluids are flowed ina laminar parallel manner across the device to two outlets in an outletportion of the device, wherein the laminar flowing fluids are present asnon-mixing streams or stream-tubes. As also shown in FIG. 17B, thefluids flowing across the device from the input area to the output areaencounter an array of obstacle there between. The array of obstacles canbe a DLD array (tilted post array in FIG. 17B) as provided herein. Thearray can be configured to separate particles in the laminar, parallelflowing streams based on size in a deterministic manner as describedherein. Particles larger than the critical size of the DLD array can be“bumped” as described herein toward the ‘bottom’ of the DLD array and besubsequently flowed toward and harvested from an outlet (product outportion of FIG. 17B). As shown in FIG. 17B, the two inlets are separatedfrom each other by a wall. In some cases, a wall separating adjacentinlets in a multi-inlet car wash device as provided herein extends intothe interior of a channel comprising the inlets, DLD array, and outletsuntil the wall encounters or abuts an edge of the DLD array. The edge ofthe DLD array can be the edge closest or nearest to the inlets. As shownin FIG. 17B, the edge of the DLD array is perpendicular to the wallseparating the inlets. As shown in FIG. 17B, the two outlets areseparated from each other by a wall. As shown in FIG. 17B, the wallseparating adjacent outlets in a ‘car wash’ device as provided hereincan extend from an edge of the DLD array nearest to the outlets to theend of the channel, wherein the edge of the DLD array is perpendicularto the wall separating the outlets. Based on the principles of DLD asdescribed herein, particles (e.g., cells, nucleic acids, reagents suchas antibodies, probes, etc.) above a critical size will flow in thedirection of bulk fluid flow and exit the device in one outlet (wasteoutput on FIG. 17B), while particles (e.g., cells, nucleic acids, etc.)above the critical size exit the device from a second or separate outlet(product out in FIG. 17B). As shown in FIG. 17B, a ‘car wash’ device asprovided herein is used to wash white blood cells (e.g., leukocytes) ina sample comprising binding agents comprising labels (e.g., labelingmolecules) and smaller cells {e.g., RBCs). As can be seen, the sample isflowed from the input area across the DLD array to the output area,wherein upon entering the tilted post array, the white blood cells arebumped from the sample flow stream into the parallel buffer flow stream,while the binding agents comprising labels (e.g., labeling molecules)and RBCs remain in the sample stream. The white blood cells can then beharvested from the product outlet essentially washed and purified fromthe labeling molecules and RBCs (FIG. 17C).

As shown in FIG. 19, a “car wash” device as provided herein can comprisea plurality of inlets and a plurality of outlets with an array ofobstacles there between (e.g., tilted post array in FIG. 19). Theplurality of inlets can be more than two inlets. The plurality ofoutlets can be more than two outlets. The device can comprise a channelbounded by a first wall and a second wall, wherein the second wallopposes the first wall, and wherein the first and second walls areconfigured to confine fluids flowing there between. The device canfurther comprise a plurality of inlets between the first and secondwall. As shown in FIG. 19, the inlets can be adjacent to each other andcan be separated by a wall. The separator walls between adjacent inletscan extend into the channel for a distance. The distance can be to thenearest perpendicular edge of the DLD array. The channel can beconfigured to flow a plurality of fluids in streams from the pluralityof inlets across the DLD array to the plurality of outlets. The streamscan be flowed in a parallel, laminar manner, wherein adjacent flowingstreams (‘flow streams’ or ‘streamtubes’) experience minimal (e.g., dueto limited diffusion), no or substantially no mixing between theadjacent flow streams. In some cases, a particle (e.g., cell) can moveat an angle to the parallel flow streams by the action of deterministiclateral displacement (DLD). In some cases, cells are introduced withouta processing chemical or enzymatic reagent, moved into a flow streamcomprising said chemical or enzymatic reagent by DLD, and then out ofthe stream into a stream comprising a clean (i.e., reagent-free) buffer,and then out of a product outlet, already washed. The DLD array betweenthe plurality of inlets and outlets can be any DLD array as providedherein. In some cases, the DLD array comprises round microposts. In somecases, the DLD array comprises triangular microposts. In some cases, theDLD comprises both round and triangular microposts. The microposts canhave any of the dimensions as provided herein. The fluid streams can beflowed through the channel at any of the flow rates as provided herein.In some cases, a car wash device as provided herein is configured to behigh-throughput, wherein fluid streams are flowed at high flow speeds(e.g., at least 1 ml/min). In some cases, a car wash device as providedherein is configured to or adapted to be high-throughput, wherein avolume of sample for running through the device is greater than a 100mls. FIG. 19 shows a device comprising 3 inlets in the input area of adevice comprising a DLD array. The 3 inlets are configured to flow 3flow streams parallel to each other. A first flow stream comprises asample, wherein the sample comprises particles, a second flow streamcomprises a reagent, and a third flow stream comprises a wash buffer.The device is configured such that the sample is inputted into theinlets nearest a first boundary wall and particles within the sample aredeflected toward a second, opposing boundary wall as the particles movethrough the DLD array, whereby the particles within the sample areseparated by size in a deterministic manner. Particles within the sampleabove a critical size of the DLD array can be deflected toward thesecond wall, while particles below the critical size can flow throughthe DLD array in the direction of the flow streams. In FIG. 19,particles within the sample stream deflected toward the second wall passthrough the reagent stream, and then the buffer flow stream, wherein areagent within the reagent flow stream can react with the particles asthey pass through the reagent stream, and wherein the reagent can bewashed off or removed from the particles as they flow through thesubsequent buffer flow stream. As shown in FIG. 19, the particlesdeflected toward the second wall can be concentrated as they encounterthe second wall, and can be subsequently flow through and harvested fromone (e.g., product in FIG. 19) of the plurality of outlets in the outputarea of the device, while particles not deflected toward the second wallas well as reagent from the reagent stream flows through one or moreoutlets of the plurality of outlets that are separate from the productoutlet (waste outlet in FIG. 19). The deflected particles collected froma device as provided can be free or substantially free of reagentsfollowing flow of the particles through a reagent stream in amulti-stream, ‘car wash’ device as provided herein.

FIG. 18A shows an embodiment of a ‘car wash’ device as described herein.The device in FIG. 18A comprises a plurality of inlets and a pluralityof outlets with an array of obstacles (tilted post array in FIG. 18A)disposed there between. The plurality of inlets can be configured toflow a plurality of flow streams toward the plurality of outlets,wherein the plurality of flow streams each comprises a separate fluid.In FIG. 18A, the channel comprises 6 inlets configured to flow sixseparate flow streams in laminar, flow streams across a tilted postarray toward 2 outlets, a product outlet, and a waste outlet. A firstflow stream comprises a sample comprising particles, a second streamcomprises a buffer, a third stream comprises a fix and permabilizationstream, a fourth stream comprises a buffer, a fifth stream comprises anintracellular label stream, and a sixth stream comprises a buffer. Insome cases, a multi-stream device as described herein comprises aplurality of parallel flow streams flowing from an input portion of thedevice to an output portion of the device, wherein at least 4 of theflow streams comprise a reagent. The at least 4 flow streams comprisinga reagent can comprise the same and/or different reagent. In some cases,each of the flow streams comprising a reagent is bounded by two parallelflow streams, each of which carries a buffer. The buffer can be a washbuffer. A device as depicted in FIG. 18A can be configured to deflectparticles (e.g., leukocytes in a sample comprising leukocytes and RBCs,e.g., blood) of a predetermined size (e.g., above a critical size of thetilted post array) from the sample stream through the subsequent fiveparallel flow streams in series (e.g.,sample→buffer→fix/perm→buffer→intracell. Label→buffer in FIG. 18A). Thebuffer streams can serve to wash reagents adsorbed non-specifically(i.e., weakly) to the particles (e.g., cells) and unbound reagents fromthe preceding adjacent flow stream from the environment of the particlesdeflected through the streams. The buffer stream can remove orsubstantially remove non-specifically bound and unbound reagent from aparticle as well as from the stream comprising the particles. As shownin FIG. 18A, a waste outlet can have a width that is greater than thewidth of the product outlet. The waste outlet in FIG. 18A comprisesreagents (e.g., surface labeling Mabs, fix/perm reagents, andintracellular binding agents comprising a label as well as undesiredparticles, e.g., RBCs, below the critical size of the tilted postarray).

F. “Car Wash” Device with Separator Walls

A multi-stream (e.g., ‘car wash’) device as provided herein can furthercomprise a mechanism to reduce mixing between laminar, parallel flowingfluids or flow streams. Mixing between parallel flow streams can occurover time, which can contaminate the output of a device as providedherein. As such, a tradeoff can exist between long processing times thatcan be required for some chemical or enzymatic streams and having lowcontamination in a final output of a car wash device as provided herein.For example, incubation times for fixation and/or permeabilization canbe ˜10 min, while labeling with a labeling agent (e.g., antibodies,probes) can be ˜5 min. In comparison, Table 1 shows diffusioncoefficients of some reagents.

TABLE 1 Diffusion Coefficients of Common Reagents Diffusion Coefficient(in Reagents water) Functionality Monoclonal Anitbodies 10⁻⁷-10⁻⁸cm²/sec Surface Labeling (mAbs) RBC lysis buffer N/A LysisParaformaldehyde (PFA) ~−10⁻⁸ cm²/sec Fixation Ethanol 10⁻⁵ cm²/secFixation Methanol 10⁻⁵ cm²/sec Fixation and permeabilization Acetone10⁻⁵ cm²/sec Fixation and permeabilization Saponin ~10⁻⁶ cm²/secPermeabilization Triton X-100 ~10⁻⁶ cm²/sec Permeabilization Tween 20~10⁻⁷ cm²/sec Permeabilization

Reagents with relatively small diffusion coefficients (e.g. methanol),can diffuse into adjacent streams even with short incubation times,which can lead to a significant percentage of the reagent(s) entering aproduct stream as can be predicted by a limited source diffusion model(FIG. 22). In some cases, particle flow angle can be adjusted withrespect to the horizontal flow streams. In some cases, a separator wallis used to separate adjacent flow streams in a device as providedherein. In some cases, a multi-stream device as provided hereincomprises one or more separator walls. In some cases, a multi-streamdevice as provided herein comprises a plurality of separator walls. Theseparator walls can be in pairs. In some cases, one of the pair ofseparator walls extends from an input portion of a channel comprisingthe pair of separator walls, while the other of the pair extends from anoutlet portion of the channel. In some cases, a separator wall or pairof separator walls are parallel to the stream flow and do not interferewith the stream flow patterns. The pair of separator walls can beopposing. As shown in FIGS. 24A and B, the pair of separator walls canbe staggered with respect to each other, whereby one of the pair iscloser to a first wall that bounds the channel, while the other of thepair is closer to a second wall that bounds the channel. In some cases,a pair of separator walls comprises a gap between separator walls in thepair of separator walls (e.g., FIGS. 24A and B). The gap can comprise awidth such that particles can pass through the gap. In some cases, amulti-stream device as provided herein comprises a plurality ofseparator wall pairs (e.g., FIG. 32). As depicted in FIG. 32, each ofthe pair of separator walls can be staggered and can comprise a gapbetween the pair that is configured to allow particles being deflectedthrough an array of obstacles (e.g., DLD array) to pass between the pairof separator walls. In some cases, a separator wall or pair of separatorwalls is placed between a reagent stream (e.g., chemical or enzyme) andan adjacent buffer (e.g., wash buffer) stream.

A separator wall or pair of separator walls in a device as providedherein can serve to substantially increase the amount of time a particleresides in a flow stream (e.g., reagent). The increase in time aparticle resides in a flow stream can be increased by at least, at most,less than, more than, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% ofthe time a particle would spend in a flow stream (e.g., reagent flowstream) in a device as provided herein that does not comprise aseparator wall or pair of separator walls.

A separator wall or pair of separator walls can serve to substantiallylimit or block the diffusion of a reagent (e.g., chemical and/orenzymatic) to an adjacent flow stream in a multi-stream (e.g., car wash)device as provided herein. The diffusion can be limited by at least, atmost, less than, more than, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%.

In some cases, a flow stream in a multi-stream (e.g., car wash) deviceas provided herein comprises a lysis reagent. In some cases, a lysisreagent comprises a detergent. In some cases, a detergent comprisesTriton X-100, SDS, CHAPs, or Tween-20.

In some cases, a flow stream in a multi-stream (e.g., car wash) deviceas provided herein comprises a buffer. The buffer can be a wash buffer.The buffer can be F108. N-(2-Acetamido)-2-aminoethanesulfonic acid(ACES); N-(2-Acetamido)iminodiacetic acid (ADA); Magnesium acetate;Sodium acetate; Trizma® base; 2-Amino-2-methyl-1-propanol (AMP);Aminoacetic acid; Aminoethanoic acid; 2-Amino-2-methyl-1,3-propanediol(AMPD); Ammonium phosphate monobasic; Ammonium sodium phosphate dibasictetrahydrate; Ammonium sodium phosphate dibasic tetrahydrate; Ammoniumbicarbonate; Ammonium phosphate monobasic; Sodium5,5-diethylbarbiturate; N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonicacid (BES); Bis(2-hydroxyethyl)amine, 2,2′-Iminodiethanol(Diethanolamine);2-Bis(2-hydroxyethyl)amino-2-(hydroxymethyl)-1,3-propanediol;Bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane;N,N-Bis(2-hydroxyethyl)glycine; N,N-Bis(2-hydroxyethyl)taurine;2,2-Bis(hydroxymethyl)-2,2′,2″-nitrilotriethanol; BIS-TRIS; Calciumacetate hydrate; Calcium carbonate; Calcium citrate tribasictetrahydrate; Calcium formate; CAPS;N-(Carbamoylmethyl)-2-aminoethanesulfonic acid;-(Carbamoylmethylamino)ethanesulfonic acid;N-(Carbamoylmethyl)iminodiacetic acid; N-(Carbamoylmethyl)taurine; CHES;Citric acid; 3-(Cyclohexylamino)-1-propanesulfonic acid; MOPS; HEPES;Imidazole; HEPPS; Formic acid; Glycine; EPPS; Edetate disodium saltdehydrate; Magnesium acetate; Lithium acetate; Oxalic acid; Phosphatebuffered saline; Piperazine; PIPES; Potassium bicarbonate; Potassiumcarbonate; Potassium chloride; lithium chloride; Potassium phosphate;propionic acid; Sodium acetate; Sodium bicarbonate; Sodium carbonate;Sodium citrate; Sodium phosphate; Sodium tetraborate; STE buffersolution; STET buffer solution; TRIS buffered saline; TRIS-EDTA buffersolution; Sodium pyrophosphate tetrabasic; Trizma® carbonate; Trizma®hydrochloride; Trizma® maleate; TRIS NaCl Tween 20; Triethanolamine;Trizma® acetate; and/or TAPS.

In some cases, a flow stream in a multi-stream (e.g., car wash) deviceas provided herein comprises a reagent. The reagent can be a chemical orenzymatic reagent. The reagent can be a fixative, permeabilizationagent, enzyme, cleavage agent, cytotoxic agent, small molecule, drugmoiety, chemotherapeutic agent or a combination or mixture thereof.

In some cases, the reagent is a fixative. The fixative can beformaldehyde, gluteraldehyde, methylalcohol, and/or ethylalcohol. Thefixative can be phosphate buffered formalin; formal calcium; formalsaline; zinc formalin (unbuffered); Zenker's fixative; Helly's fixative;B-5 fixative; Bouin's solution; Hollande's; Gendre's solution; Clarke'ssolution; Carnoy's solution; Methacarn; Alcoholic formalin; and/orformol acetic alcohol.

In some cases, the reagent is a permeabilization agent. Thepermeabilization agent can be detergents, alcohols (methylalcohol),membrane disrupting toxics like digitonin, melittin, and/or saponin. Thedetergents can be 2-aminoethyl methan thiosulfonate hydrobromide, CHAPS,CHAPSO, digitonin, lithium dodecyl sulfate,n-dodecyl-beta-D-maltopyranoside, n-octyl-beta-d-glucopyranoside,NDSB-195, NDSB-201, NDSB-211, NDSB-221, NDSB-256, NONIDET-P40, PluronicF68, Pluronic F-127, MTSES, Tween-20, Tween-80, Tween-40, sodium dodecylsulfate (SDS), Triton X-100, Triton X-114, MTSET, sulfobetaine-10,sulfobetaine-12, or sulfobetaine-14, Igepal® CA-630, orn-dodecyl-β-D-maltoside (DDM).

In some cases, the reagent comprises a binding agent. A reagentcomprising a binding agent can also be referred to as a labeling agent.The labeling agent can be any labeling agent known in the art. Thelabeling agent can be a cell surface labeling agent. The labeling agentcan be an intracellular labeling agent. The labeling agent can beantibodies, antibody fragments, nucleic acid probes, aptamers, molecularbeacons, and/or enzyme substrates. The binding agent can be an antibody,antibody fragment, a nucleic acid (e.g., probe), aptamer, smallmolecule, or molecular beacon. The antibody can be a primary orsecondary antibody. The term “antibody” herein can be used in thebroadest sense and specifically covers monoclonal antibodies, polyclonalantibodies, multispecific antibodies, and antibody fragments so long asthey exhibit the desired biological activity. The term “multispecificantibody” can be used in the broadest sense and specifically covers anantibody comprising an antigen-binding domain that has polyepitopicspecificity (i.e., is capable of specifically binding to two, or more,different epitopes on one biological molecule or is capable ofspecifically binding to epitopes on two, or more, different biologicalmolecules). One specific example of an antigen-binding domain is aV_(H)V_(L) unit comprised of a heavy chain variable domain (V_(H)) and alight chain variable domain (V_(L)). Such multispecific antibodies caninclude, but are not limited to, full length antibodies, antibodieshaving two or more V_(L) and V_(H) domains, antibody fragments such asFab, Fv, dsFv, scFV, diabodies, bispecific diabodies and triabodies,antibody fragments that have been linked covalently or non-covalently. A“bispecific antibody” can be a multispecific antibody comprising anantigen-binding domain that can be capable of specifically binding totwo different epitopes on one biological molecule or can be capable ofspecifically binding to epitopes on two different biological molecules.The bispecific antibody can also be referred to as having “dualspecificity” or as being “dual specific”. In some cases, the bindingreagent is a nucleic acid probe, wherein the nucleic acid probecomprises one or nucleotides comprising a label incorporated into thenucleic acid probe. The nucleic acid probe can be comprise DNA, RNA, ora combination thereof. The labeled nucleotides can be labeled with AlexaFluor® dyes. Other fluorescent labels on nucleotides for use in nucleicacid probes can be, but are not limited to, Diethylaminocoumarin (DEAC),Cyanine 3 (Cy3), Cyanine 5 (Cy5), Fluorescein (FITC), Lissamine, R110,R6G, Tetramethylrhodamine (TAMRA) and Texas Red. The labeled nucleotidescan be labeled with a hapten. The hapten label can be, but are notlimited to Amino-digoxigenin (DIG), Biotin, Dinitrophenyl (DNP) andFluorescein (FITC). The labeled nucleotides can comprise a radioactivelabel. For example, radioactive labeled nucleotides can include, but arenot limited to, ³³P, ³²P, ³⁵S, ³H and ¹⁴C nucleotides. In some cases,the binding agent binds or is directed to or against a cell surfacemarker. The cell surface marker can be any cell surface marker asprovided herein. In some cases, the binding agent binds or directed toor against an intracellular marker. The intracellular marker can be anyintracellular marker as provided herein. In some cases, a device asprovided herein is adapted to flow a reagent flow stream comprising abinding agent, wherein the binding agent binds a cell surface marker. Insome cases, a device as provided herein is adapted to flow a reagentflow stream comprising a binding agent, wherein the binding agent bindsan intracellular marker. In some cases, a device as provided herein isadapted to flow a plurality of flow streams, wherein at least one of theflow streams comprises a binding agent that binds a cell surface marker,and at least one of the flow streams comprises a binding agent thatbinds an intracellular marker. In some cases, a device as providedherein comprises a plurality of flow streams, wherein at least of theflow streams comprises a reagent, wherein the reagent is a bindingagent, and wherein at least of the flow streams comprises a reagent thatis not a binding agent. The reagent that is not binding agent can be anenzyme, a fixative, a permeabilization agent or a combination or mixturethereof.

In some cases, the reagent comprises an enzyme. In some cases, thereagent comprises a plurality of enzymes. In some cases, a device asprovided herein comprises a plurality of flow streams, wherein at leastof the flow streams comprises a reagent, wherein the reagent is anenzyme. In some cases, a device as provided herein comprises a pluralityof flow streams, wherein at least of the flow streams comprises areagent, wherein the reagent is an enzyme, and wherein at least of theflow streams comprises a reagent that is not an enzyme. The reagent thatis not an enzyme can be a binding agent, a fixative, a permeabilizationagent or a combination or mixture thereof. In some cases, a device asprovided herein comprises a plurality of flow streams, wherein more thanone of the flow streams comprises a reagent, wherein the reagent in eachof the more than one flow streams is an enzyme, wherein the enzyme ineach of the more than one flow streams comprises the same or differentenzyme. In some cases, a device as provided herein is adapted to flow aplurality of flow streams, wherein at least one of the flow streamscomprises a binding agent that binds a cell surface marker, at least oneof the flow streams comprises a binding agent that binds anintracellular marker, and at least one of the flow streams comprises anenzyme. The enzyme can be a restriction enzyme, protease, polymerase,ligase, nuclease, endonuclease, exonuclease, phosphatase, methylase,topoisomerase or a combination or mixture thereof. The polymerase can beany polymerase known in the art. The polymerase can be a DNA dependentDNA Polymerase. Examples of DNA-dependent DNA polymerase include, butare not limited to, Klenow polymerase, with or without 3′-exonuclease,Bst DNA polymerase, Bca polymerase, .phi.29 DNA polymerase, Ventpolymerase, Deep Vent polymerase, Taq polymerase, T4 polymerase, and E.coli DNA polymerase 1, derivatives thereof, or mixture of polymerases.In some cases, the polymerase does not comprise a 5′-exonucleaseactivity. In other cases, the polymerase comprises 5′ exonucleaseactivity. The polymerase can be a RNA dependent DNA Polymerase orreverse transcriptase (RT). Examples of RTs include, but are not limitedto, Moloney murine leukemia virus (M-MLV) reverse transcriptase, humanimmunodeficiency virus (HIV) reverse transcriptase, rous sarcoma virus(RSV) reverse transcriptase, avian myeloblastosis virus (AMV) reversetranscriptase, rous associated virus (RAV) reverse transcriptase, andmyeloblastosis associated virus (MAV) reverse transcriptase or otheravian sarcoma-leukosis virus (ASLV) reverse transcriptases, and modifiedRTs derived therefrom. Exonuclease can be, but are not limited toexonuclease 1, exonuclease 7 or a combination or mixture thereof.Endonucleases can be, for example, but not limited to mung beanendonuclease or Si endonuclease or a combination or mixture thereof.

In some cases, the reagent comprises a label. In some cases, the reagentcomprises a binding agent, wherein the binding agent comprises a label.In some cases, the reagent comprises an enzyme, wherein the enzymecomprises a label. The label can be conjugated, bound or linked to areagent as provided herein. The label can refer to any atom or moleculeknown in the art that can be used to provide a detectable and/orquantifiable effect. The label can be attached to a nucleic acid orprotein (e.g., antibody, antibody fragment, and/or enzyme). In somecases, the label provides a quantifiable effect. In some cases, thelabel provides a detectable and quantifiable effect. Labels can includebut are not limited to dyes; radiolabels such as ³²P; binding moietiessuch as biotin; haptens such as digoxgenin; luminogenic, phosphorescentor fluorogenic moieties; mass tags; and fluorescent dyes alone or incombination with moieties that can suppress or shift emission spectra byfluorescence resonance energy transfer (FRET). Labels can providesignals detectable by fluorescence, radioactivity, colorimetry,gravimetry, X-ray diffraction or absorption, magnetism, enzymaticactivity, characteristics of mass or behavior affected by mass (e.g.,MALDI time-of-flight mass spectrometry), and the like. A label can be acharged moiety (positive or negative charge) or alternatively, can becharge neutral. In some cases, the label is a fluorescent dye. Thefluorescent dye can be squaric acid-based dyes. The squaric acid-baseddyes can be selected from cyclobutenedione derivatives, symmetrical andunsymmetrical squaraines, substituted cephalosporin compounds,fluorinated squaraine compositions, alkylalkoxy squaraines, orsquarylium compounds. The squaric acid-based dyes can be selected from ared fluorescent dye and an orange fluorescent dye, such as the redfluorescent dye comprising1,3-bis(1,3-dihydro-1,3,3-trimethyl-2H-indol-2-ylidene)methyl]-2,4-dihydr-oxycyclobutenediylium, bis(inner salt) and the orange fluorescent dyecomprising2-(3,5-dimethylpyrrol-2-yl)-4-(3,5-dimethyl-2H-pyrrol-2-ylidene)-3-hydrox-y-2-cyclobuten-1-one.Labels can include or consist of nucleic acid or protein sequence, solong as the sequence comprising the label is detectable and/orquantifiable.

In some cases, the reagent comprises a cytotoxic agent, toxin, orchemotherapeutic agent. For example, the cytotoxic agent or toxin canbe, but is not limited to, enzymatically active toxins and fragmentsthereof including diphtheria A chain, nonbinding active fragments ofdiphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricinA chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, or the tricothecenes, or combinations or mixtures thereof.Examples of small molecule toxins can include, but are not limited to, acalicheamicin, maytansinoids, dolastatins, aurostatins, a trichothecene,or CC 1065, or the derivatives of these toxins that have toxin activity.Examples of chemotherapeutic agents include, but are not limited toalkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®);alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol(dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinicacid; a camptothecin (including the synthetic analogue topotecan(HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin,scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); podophyllotoxin; podophyllinic acid; teniposide;cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogues, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e. g.,calicheamicin, especially calicheamicin gamma 1I and calicheamicinomegall (see, e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33:183-186 (1994)); CDP323, an oral alpha-4 integrin inhibitor; dynemicin,including dynemicin A; an esperamicin; as well as neocarzinostatinchromophore and related chromoprotein enediyne antibiotic chromophores),aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins,dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,doxorubicin (including ADRIAMYCIN®, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HClliposome injection (DOXIL®), liposomal doxorubicin TLC D-99 (MYOCET®),peglylated liposomal doxorubicin (CAELYX®), and deoxydoxorubicin),epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such asmitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin,porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;anti-metabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur(UFTORAL®), capecitabine (XELODA®), an epothilone, and 5-fluorouracil(5-FU); folic acid analogues such as denopterin, methotrexate,pteropterin, trimetrexate; purine analogs such as fludarabine,6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such asancitabine, azacitidine, 6-azauridine, carmofur, cytarabine,dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens suchas calusterone, dromostanolone propionate, epitiostanol, mepitiostane,testolactone; anti-adrenals such as aminoglutethimide, mitotane,trilostane; folic acid replenisher such as frolinic acid; aceglatone;aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine;bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS NaturalProducts, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2′-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A andanguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); thiotepa; taxoid, e.g., paclitaxel (TAXOL®),albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANE™),and docetaxel (TAXOTERE®); chloranbucil; 6-thioguanine; mercaptopurine;methotrexate; platinum agents such as cisplatin, oxaliplatin (e.g.,ELOXATIN®), and carboplatin; vincas, which prevent tubulinpolymerization from forming microtubules, including vinblastine(VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), andvinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone;leucovorin; novantrone; edatrexate; daunomycin; aminopterin;ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid, including bexarotene(TARGRETIN®); bisphosphonates such as clodronate (for example, BONEFOS®or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronicacid/zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate(AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®);troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisenseoligonucleotides, particularly those that inhibit expression of genes insignaling pathways implicated in aberrant cell proliferation, such as,for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor(EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines,for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID®vaccine; topoisomerase 1 inhibitor (e.g., LURTOTECAN®); rmRH (e.g.,ABARELIX®); BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib, SUTENT®,Pfizer); perifosine, COX-2 inhibitor (e.g. celecoxib or etoricoxib),proteosome inhibitor (e.g. PS341); bortezomib (VELCADE®); CCI-779;tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitor such asoblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors (seedefinition below); tyrosine kinase inhibitors (see definition below);serine-threonine kinase inhibitors such as rapamycin (sirolimus,RAPAMUNE®); farnesyltransferase inhibitors such as lonafarnib (SCH 6636,SARASAR™); and pharmaceutically acceptable salts, acids or derivativesof any of the above; as well as combinations of two or more of the abovesuch as CHOP, an abbreviation for a combined therapy ofcyclophosphamide, doxorubicin, vincristine, and prednisolone; andFOLFOX, an abbreviation for a treatment regimen with oxaliplatin(ELOXATIN™) combined with 5-FU and leucovorin. Additionalchemotherapeutic agents can include “anti-hormonal agents” or “endocrinetherapeutics” which act to regulate, reduce, block, or inhibit theeffects of hormones that can promote the growth of cancer. They can behormones themselves, including, but not limited to: anti-estrogens withmixed agonist/antagonist profile, including, tamoxifen (NOLVADEX®),4-hydroxytamoxifen, toremifene (FARESTON®), idoxifene, droloxifene,raloxifene (EVISTA®), trioxifene, keoxifene, and selective estrogenreceptor modulators (SERMs) such as SERM3; pure anti-estrogens withoutagonist properties, such as fulvestrant (FASLODEX®), and EM800 (suchagents may block estrogen receptor (ER) dimerization, inhibit DNAbinding, increase ER turnover, and/or suppress ER levels); aromataseinhibitors, including steroidal aromatase inhibitors such as formestaneand exemestane (AROMASIN®), and nonsteroidal aromatase inhibitors suchas anastrazole (ARIMIDEX®), letrozole (FEMARA®) and aminoglutethimide,and other aromatase inhibitors include vorozole (RIVISOR®), megestrolacetate (MEGASE®), fadrozole, and 4(5)-imidazoles; lutenizinghormone-releasing hormone agonists, including leuprolide (LUPRON® andELIGARD®), goserelin, buserelin, and tripterelin; sex steroids,including progestines such as megestrol acetate and medroxyprogesteroneacetate, estrogens such as diethylstilbestrol and premarin, andandrogens/retinoids such as fluoxymesterone, all transretionic acid andfenretinide; onapristone; anti-progesterones; estrogen receptordown-regulators (ERDs); anti-androgens such as flutamide, nilutamide andbicalutamide; and pharmaceutically acceptable salts, acids orderivatives of any of the above; as well as combinations of two or moreof the above.

In some cases, particles processed using a multi-stream device asprovided herein can be processed by chemical and/or enzymatic reactionswithin the bump array to impart fluorescent, magnetic, or radioactiveproperties to these molecules.

G. Device Features

As described herein, a multi-stream (e.g., ‘car wash’) device asprovided herein can comprise a channel with a plurality of inlets, aplurality of outlets, and an array of obstacles disposed there between.Exemplary devices can be the devices illustrated in FIGS. 18A-B, 19,20A-B, 24A-B, 27A-B, and 32. In some cases, a device as provided hereincan comprises a channel with at least one input, at least one output,and an array of obstacles disposed there between. Exemplary devices canbe the devices illustrated in FIGS. 39 and 40. Examples of parameters ofdevices are illustrated in Table 2.

TABLE 2 Channel widths A/A2 B C Blood inlet channel width (μm) 50 100150 Buffer inlet channel width (μm) 55 110 110 Product outlet channelwidth (μm) 49 98 98

TABLE 3 Gap size (edge-to-edge distance between posts)/post diameter(μm) A B C Section 1 18/27 44/66  90/135 Section 2 12/18 30/45 60/90Section 3  8/12 20/30 40/60

FIG. 39 shows a design of an A chip. The A chip comprises a three zone(section) design with progressively smaller pillars and gaps. Gap sizeand post diameter are described in Table 2. The device can comprise aninlet, e.g., for blood, an inlet for buffer, waste outlets, and aproduct outlet. The A chip can comprise 14 parallel channels. The totalchannel volume (including 0.5 mm vias) (a via can be a hole that canconnect the backside of a chip, (e.g., where the manifold connection canoccur) to the top side of the chip (i.e., where the array is located)can be 118 μL. The throughput of the device is about 4-8 mL/hr. Theprocessing time for an 8 mL sample can be about 1 to about 2 hours. TheA chip can be made with silicon. The A chip can be made withpolypropylene, poly(methyl methacrylate) (PMMA), or cyclo-olefin polymer(COP). In some cases, between about 5 mL and about 20 mL of sample canbe applied to the device. In some cases, between about 1 μL and about 5mL of sample can be applied to the device.

FIG. 40 shows another example of a device (A2). The A2 chip comprises athree zone design with progressively smaller pillars and gaps. Gap sizesand post diameters are described in Table 3. The depth of the channel is60 μm. Each A2 chip comprises 2 independent channels. The total channelvolume (including 0.5 mm via) is about 3.85 μL. The throughput for thedevice can be about 0.12 to about 0.24 mL/hr, or about 0.4 to about 0.8mL/hr. The processing time for a 100 μL sample can be about 25 to about50 min. The A2 chip can be made with silicon. The A2 chip can be madewith polypropylene, poly(methyl methacrylate) (PMMA), or cyclo-olefinpolymer (COP). The device can be used to process about 50 to about 500uL of sample. The device can be used to process about 1 to about 50 uLof sample.

i. Channels

In some cases, a device as provided herein comprises a channel, whereinthe channel is bounded by a first wall and a second wall, wherein thesecond wall opposes the first wall, and wherein the first and secondwall are parallel to each other. The channel can comprise an inputportion or area comprising a plurality of inlets and an output portionor area comprising a plurality of outlets. Each of the plurality ofinlets can be configured to flow or allow passage of a flow stream orstream tube comprising a fluid, wherein each of the flow streams orstream tubes flows parallel to each other flow stream or stream tubefrom the input portion of the device to the outlet portion of thedevice. In some cases, each of a plurality of flow streams moves from aninlet to an outlet directly across from or opposing the inlet. In somecases, a device comprises a channel with at least one inlet and at leastone outlet. In some cases, a device comprises a channel with two inletsand two outlets. In some cases, a device comprises a channel with morethan two inlets and more than two outlets. In some cases, a first inletto a channel is a sample inlet and a second inlet to a channel is abuffer inlet. In some cases, a first inlet to a channel is a sampleinlet, a second inlet to a channel is a reagent inlet, and a third inletis a buffer inlet. A single flow stream can originate from a singleinlet. A single flow stream can originate from two or more adjacentinlets (e.g., FIG. 19). In some cases, a first outlet to a channel is aproduct outlet and a second outlet to a channel is a waste outlet. Insome cases, a first outlet of a plurality of outlets to a channel is aproduct outlet and each of the remaining plurality of outlets comprisesa waste outlet. Each of the remaining plurality of outlets can comprisea distinct outlet for a flow stream originating from an inlet present inan input portion of a device as provided herein comprising the inlet andthe waste outlet, wherein the inlet is directly opposed to or acrossfrom the waste outlet. In some cases, a channel comprises an array ofobstacles between a plurality of inlets and a plurality of outlets. Insome cases, the array of obstacles comprises zones or sections ofobstacles, wherein each section comprises obstacles of substantially thesame diameter and size and gaps between obstacles of substantially thesame size.

(a) Channel Width

In some cases, channel width is about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, or 100 mm.

In some cases channel width is at least 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, or 100 mm.

In some cases, channel width is less than 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, or 100 mm.

In some cases, channel width is about 1 to about 10 mm, about 2 to about9 mm, about 3 to about 8 mm, about 10 to about 20 mm, about 20 to about30 mm, about 30 to about 40 mm, about 40 to about 60 mm, about 60 toabout 70 mm, or about 70 to about 100 mm.

(b) Channel Length

In some cases, channel length is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mm.

In some cases, channel length is less than 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mm.

In some cases, channel length is at least 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mm.

In some cases, channel length is about 1 to about 10 mm, about 2 toabout 9 mm, about 3 to about 8 mm, about 10 to about 20 mm, about 20 toabout 30 mm, about 30 to about 40 mm, about 40 to about 60 mm, about 60to about 70 mm, about 70 to about 100 mm, or about 100 to about 200 mm.

(c) Channel Depth

In some cases, a channel has a depth of about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 μm.

In some cases, a channel has a depth of at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000μm.

In some cases, a channel has a depth less than 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000μm.

In some cases, a channel has a depth of about 10 to about 30 μm, about20 to about 40 μm, about 30 to about 50 μm, about 50 to about 100 μm,about 100 to about 200 μm, about 200 to about 400 μm, about 400 to about600 μm, or about 600 to about 1000 μm.

(d) Number of Channels Per Device (Chip)

In some cases, a device comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or100 channels. In some cases, a device comprises at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, or 100 channels.

In some cases, a device comprises about 2 to about 10 channels, about 10to about 20 channels, about 20 to about 30 channels, about 30 to about40 channels, about 40 to about 50 channels, about 50 to about 60channels, about 60 to about 70 channels, or about 70 to about 100channels.

(e) Channel Volume

In some cases, a total volume of a channel is about 0.001, 0.005, 0.01,0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 150, or 200 mL.

In some cases, a total volume of a channel is at least 0.001, 0.005,0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 150, or 200 mL.

In some cases, a total volume of a channel is about 0.001 to about 0.01mL, about 0.01 to about 0.1 mL, about 0.1 to about 0.5 mL, about 1 toabout 2 mL, about 2 to about 3 mL, about 3 to about 5 mL, about 5 toabout 10 mL, about 10 to about 20 mL, or about 20 to about 50 mL.

In some cases a device comprises multiple channels. In some cases, atotal volume of the channels in a device is any of the volumes listedabove multiplied by the number of channels in the device.

In some cases, device as provide herein is adapted to process as low as˜10 μl and as high as 500 μl. In some cases, a device as provided hereinis adapted to process between 500 μl and 20 or 40 ml. In some cases, adevice as provided herein is adapted to process between more than 40 ml.

(f) Zones (Stages) within a Channel

A device described herein can have a plurality of zones (stages, orsections). A zone can be an area on a device with the same or similarsized post (obstacles) and gaps. In some cases, a channel in a devicecomprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 zones. In some cases, achannel in a device comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10zones.

In some cases, a sample, e.g., a biological sample, can compriseparticles with a broad range of sizes. If a particle in a sample islarger than a gap, the particle can clog the channel. In some cases,multiple separation stages with different gap and post sizes can beused. In some cases, post diameter and gap size is smaller in a secondzone relative to a first zone. In some cases, a device comprises aplurality of zones, wherein when a fluid is applied to an inlet of thedevice, it flows through a plurality of zones in a channel, e.g., atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 zones. In some cases, postdiameter and/or gap sizes get progressively smaller as a fluid flowsfrom and inlet to an outlet across zones in a channel.

(g) Gap Size (Edge-to-Edge Distance Between Posts or Obstacles)

In some cases, gap size in an array of obstacles (edge-to-edge distancebetween posts or obstacles) is about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5,5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13,13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200,1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400,2500, 2600, 2700, 2800, 2900, or 3000 μm.

In some cases, gap size in an array of obstacles is at least 1, 1.5, 2,2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5,11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5,18, 18.5, 19, 19.5, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175,200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900,2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, or 3000 μm.

In some cases, gap size in an array of obstacles is less than 1, 1.5, 2,2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5,11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5,18, 18.5, 19, 19.5, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175,200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900,2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, or 3000 μm.

In some cases, gap size is about 1 μm to about 10 μm, about 10 μm toabout 20 μm, about 20 μm to about 30 μm, about 30 μm to about 40 μm,about 40 μm to about 50 μm, about 50 μm to about 60 μm, about 60 μm toabout 70 μm, about 70 μm to about 80 μm, about 80 μm to about 90 μm,about 90 μm to about 100 μm, about 100 μm to about 110 μm, about 110 μmto about 120 μm, about 120 μm to about 130 μm, about 130 μm to about 140μm, about 140 μm to about 150 μm, about 150 μm to about 160 μm, about160 μm to about 170 μm, about 170 μm to about 180 μm, about 180 μm toabout 190 μm, about 190 μm to about 200 μm, about 200 μm to about 250μm, about 250 μm to about 300 μm, about 300 μm to about 400 μm, about400 μm to about 500 μm, about 500 μm to about 600 μm, about 600 μm toabout 700 μm, about 700 μm to about 800 μm, about 800 μm to about 900μm, about 900 μm to about 1000 μm, about 1000 μm to about 1500 μm, about1500 μm to about 2000 μm, about 2000 μm to about 2500 μm, or about 2500μm to about 3000 μm.

(h) Post (Obstacle) Diameter

In some cases, post (obstacle) diameter is about 1, 1.5, 2, 2.5, 3, 3.5,4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12,12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19,19.5, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125,130, 135, 140, 145, 150, 155, 160, 170, 175, 180, 185, 190, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950,1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100,2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, or 3000 μm.

In some cases, post (obstacle) diameter is at least 1, 1.5, 2, 2.5, 3,3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5,12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5,19, 19.5, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120,125, 130, 135, 140, 145, 150, 155, 160, 170, 175, 180, 185, 190, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000,2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, or 3000 μm.

In some cases, post (obstacle) diameter is less than 1, 1.5, 2, 2.5, 3,3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5,12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5,19, 19.5, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120,125, 130, 135, 140, 145, 150, 155, 160, 170, 175, 180, 185, 190, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000,2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, or 3000 μm.

(i) Obstacle Cross-Sectional Shape

In some cases, the cross-sectional shape of a post or obstacle is acircle, triangle, square, rectangle, pentagon, hexagon, heptagon,octagon, nonagon, decagon, hendecagon, dodecagon, hexadecagon, icosagon,or star. In some cases, a triangle is an acute triangle, equiliateraltriangle, isosceles triangle, obtuse triangle, rational triangle, righttriangle (30-60-90 triangle, isosceles right triangle, Kepler triangle),or scalene triangle. In some cases, the cross-sectional shape of a postor obstacle is a quadrilateral, e.g., a cyclic quadrilateral, square,kite, parallelogram, rhombus, Lozeng, rhomboid, rectangle, tangentialquadrilateral, trapezoid, trapezium, or isososceles trapezoid. In somecases, the cross-sectional shape of a post or obstacle is a crescent,ellipse, lune, oval, Reuleauz polygon, Reuleaux triangle, lens, vesicapiscis, salinon, semicircle, tomoe, magatama, triquetra, asteroid,deltoid super ellipse, or tomahawk. In some cases, a cross-sectionalshape with a point has a sharpened point. In some cases, across-sectional shape with a point has a rounded point. In some cases, across-sectional shape with more than one point has at least one roundedpoint and at least one sharpened point.

In some cases, a post (obstacle) has a cylindrical shape.

(j) Distance of Posts (Obstacles) from an Inlet

A first row of posts can be spaced less than about 1000, 950, 900, 850,800, 750, 700, 650, 600, 550, or 500 450, 400, 350, 300, 250, 200, 150,100, 50, 40, 30, 20, 10, or 5 μm from an input.

(k) Tilt Angle

In some cases an array of obstacles has a tilt angle E (with respect tothe direction of fluid flow) of ⅓, ¼, ⅕, ⅙, 1/7, ⅛, 1/9, 1/10, 1/11,1/12, 1/13, 1/14, 1/15, 1/16, 1/17, 1/18, 1/19, 1/20, 1/21, 1/22, 1/23,1/24, 1/25, 1/26, 1/27, 1/28, 1/29, 1/30, 1/31, 1/32, 1/33, 1/34, 1/35,1/36, 1/37, 1/38, 1/39, 1/40, 1/41, 1/42, 1/43, 1/44, 1/45, 1/46, 1/47,1/48, 1/49, 1/50, 1/51, 1/52, 1/53, 1/54, 1/55, 1/56, 1/57, 1/58, 1/59,1/60, 1/61, 1/62, 1/63, 1/64, 1/65, 1/66, 1/67, 1/68, 1/69, 1/70, 1/71,1/72, 1/73, 1/74, 1/75, 1/76, 1/77, 1/78, 1/79, 1/80, 1/81, 1/82, 1/83,1/84, 1/85, 1/86, 1/87, 1/88, 1/89, 1/90, 1/91, 1/92, 1/93, 1/94, 1/95,1/96, 1/97, 1/98, 1/99, 1/100, 1/110, 1/120, 1/130, 1/140, 1/150, 1/160,1/170, 1/180, 1/190, 1/200, 1/300, 1/400, 1/500, 1/600, 1/700, 1/800,1/900, 1/1000, 1/2000, 1/3000, 1/4000, 1/5000, 1/6000, 1/7000, 1/8000,1/9000, or 1/10,000 radian.

In some cases, ε is less than ⅓, ¼, ⅕, ⅙, 1/7, ⅛, 1/9, 1/10, 1/11, 1/12,1/13, 1/14, 1/15, 1/16, 1/17, 1/18, 1/19, 1/20, 1/21, 1/22, 1/23, 1/24,1/25, 1/26, 1/27, 1/28, 1/29, 1/30, 1/31, 1/32, 1/33, 1/34, 1/35, 1/36,1/37, 1/38, 1/39, 1/40, 1/41, 1/42, 1/43, 1/44, 1/45, 1/46, 1/47, 1/48,1/49, 1/50, 1/51, 1/52, 1/53, 1/54, 1/55, 1/56, 1/57, 1/58, 1/59, 1/60,1/61, 1/62, 1/63, 1/64, 1/65, 1/66, 1/67, 1/68, 1/69, 1/70, 1/71, 1/72,1/73, 1/74, 1/75, 1/76, 1/77, 1/78, 1/79, 1/80, 1/81, 1/82, 1/83, 1/84,1/85, 1/86, 1/87, 1/88, 1/89, 1/90, 1/91, 1/92, 1/93, 1/94, 1/95, 1/96,1/97, 1/98, 1/99, 1/100, 1/110, 1/120, 1/130, 1/140, 1/150, 1/160,1/170, 1/180, 1/190, 1/200, 1/300, 1/400, 1/500, 1/600, 1/700, 1/800,1/900, 1/1000, 1/2000, 1/3000, 1/4000, 1/5000, 1/6000, 1/7000, 1/8000,1/9000, or 1/10,000 radian.

In some cases, the tilt angle is between about 1/1000 to about ⅓, orabout 1/100 to about ⅕, or about 1/1000 to about 1/100, or about 1/500to about 1/100, or about 1/50 to about ⅓.

(l) Inlet or Inlet Channel Width

In some cases, each of a plurality of inlets in a device as providedherein comprising the plurality of inlets has the same or substantiallythe same width. In some cases, each of a plurality of inlets in a deviceas provided herein comprising the plurality of inlets has a different orsubstantially different width. In some cases, one or more of a pluralityof inlets in a device as provided herein comprising a plurality ofinlets comprises a sample input, wherein the sample input has adifferent or substantially different width than each of the otherplurality of inlets. In some cases, each inlet of a plurality of inletsis an inlet channel, wherein the inlet channel is bounded by twoopposing walls. Each inlet or inlet channel can correspond to a flowstream. In some cases, an inlet (e.g., sample, buffer, and/or reagent)channel width is about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5,7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14,14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152,153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166,167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180,181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194,195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222,223, 224, 225, 230, 235, 240, 245, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, or1500 μm

In some cases, an inlet (e.g., sample, buffer, and/or reagent) channelwidth is at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7,7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5,15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181,182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209,210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223,224, 225, 230, 235, 240, 245, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, or 1500μm.

In some cases, an inlet (e.g., sample, buffer, and/or reagent) channelwidth is less than 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7,7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5,15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181,182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209,210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223,224, 225, 230, 235, 240, 245, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, or 1500μm.

In some cases, an inlet (e.g., sample, buffer, and/or reagent) channelwidth is about 1 to about 10 μm, about 10 to about 20 μm, about 20 toabout 30 μm, about 30 to about 60 μm, about 60 to about 90 μm, about 90to about 120 μm, about 120 to about 180 μm, about 180 to about 250 μm,about 250 to about 500 μm, about 500 to about 1000 μm, about 1000 toabout 1500 μm.

(m) Product Outlet or Product Outlet Channel Width

In some cases, each of a plurality of outlets in a device as providedherein comprising the plurality of outlets has the same or substantiallythe same width. In some cases, each of a plurality of outlets in adevice as provided herein comprising the plurality of outlets has adifferent or substantially different width. In some cases, one or moreof a plurality of outlets in a device as provided herein comprising aplurality of outlets comprises a sample input, wherein the sample inputhas a different or substantially different width than each of the otherplurality of outlets. In some cases, each outlet of a plurality ofoutlets is an outletchannel, wherein the outlet channel is bounded bytwo opposing walls. Each outlet or outlet channel can correspond to aflow stream. In some cases, an outlet (e.g., product, waste (e.g.,reagent, and/or buffer)) channel width is about 1, 1.5, 2, 2.5, 3, 3.5,4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12,12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19,19.5, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125,130, 135, 140, 145, 150, 155, 160, 170, 175, 180, 185, 190, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950,1000, 1100, 1200, 1300, 1400, or 1500 μm.

In some cases, an outlet (e.g., product, waste (e.g., reagent, and/orbuffer)) channel width is at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5,5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13,13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140,145, 150, 155, 160, 170, 175, 180, 185, 190, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200,1300, 1400, or 1500 μm.

In some cases, an outlet (e.g., product, waste (e.g., reagent, and/orbuffer)) channel width is less than 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5,5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13,13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140,145, 150, 155, 160, 170, 175, 180, 185, 190, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200,1300, 1400, or 1500 μm.

In some cases, an outlet (e.g., product, waste (e.g., reagent, and/orbuffer)) channel width is about 1 to about 10 μm, about 10 to about 20μm, about 20 to about 30 μm, about 30 to about 60 μm, about 60 to about90 μm, about 90 to about 120 μm, about 120 to about 180 μm, about 180 toabout 250 μm, about 250 to about 500 μm, about 500 to about 1000 μm,about 1000 to about 1500 μm.

(n) Separator Wall Length

In some cases, a separator wall extends into an array of obstacles in adevice as provided herein. The separator wall can extend for at least,at most, more than, less than or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or100% of the length of a channel that comprises the array of obstaclesand the separator wall.

In some cases, separator wall length is about 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mm.

In some cases, separator wall length is less than 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mm.

In some cases, separator wall is at least 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mm.

In some cases, separator wall is about 1 to about 10 mm, about 2 toabout 9 mm, about 3 to about 8 mm, about 10 to about 20 mm, about 20 toabout 30 mm, about 30 to about 40 mm, about 40 to about 60 mm, about 60to about 70 mm, about 70 to about 100 mm, or about 100 to about 200 mm.

(o) Separator Wall Width

In some cases, a separator wall in a device as provided herein has thesame width along the entire length of the separator wall. The separatorwall can taper, wherein a first end of the separator wall has a largerwidth than a second end of the separator wall.

In some cases, separator wall width is about 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 150, 200, 250, 300,350, 400, 450, 500, 750, or 1000 μm.

In some cases, separator wall width is at least 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 150, 200, 250, 300,350, 400, 450, 500, 750, or 1000 μm.

In some cases, separator wall width is less than 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 150, 200, 250,300, 350, 400, 450, 500, 750, or 1000 μm.

In some cases, separator wall width is about 1 to about 10 μm, about 2to about 9 μm, about 3 to about 8 μm, about 10 to about 20 μm, about 20to about 30 μm, about 30 to about 40 μm, about 40 to about 60 μm, about60 to about 70 μm, about 70 to about 100 μm, about 100 to about 250,about 250 to about 500, or about 500 to about 1000 μm.

In some cases, a predetermined size is at least 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7,7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5,15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 μm.

In some cases, a predetermined size is about 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7,7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5,15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 μm.

In some cases, a predetermined size is less than 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5,7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14,14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, or 100 μm.

(p) Gap Width Between Separator Walls

In some cases, a gap exists between adjacent separator walls in amulti-stream (e.g., ‘car wash’) device as provided herein. The gap canbe configured for particles (e.g., cells) being deflected through anarray of obstacle in a device as provided herein can pass there between.In some cases, a gap width between separator walls (e.g., opposingseparator walls) is about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6,6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14,14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152,153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166,167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180,181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194,195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222,223, 224, 225, 230, 235, 240, 245, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, or1500 μm.

In some cases, a gap width between separator walls (e.g., opposingseparator walls) is at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6,6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14,14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152,153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166,167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180,181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194,195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222,223, 224, 225, 230, 235, 240, 245, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, or1500 μm.

In some cases, a gap width between separator walls (e.g., opposingseparator walls) is less than 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6,6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14,14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152,153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166,167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180,181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194,195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222,223, 224, 225, 230, 235, 240, 245, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, or1500 μm.

In some cases, a gap width between separator walls (e.g., opposingseparator walls) is about 1 to about 10 μm, about 10 to about 20 μm,about 20 to about 30 μm, about 30 to about 60 μm, about 60 to about 90μm, about 90 to about 120 μm, about 120 to about 180 μm, about 180 toabout 250 μm, about 250 to about 500 μm, about 500 to about 1000 μm,about 1000 to about 1500 μm.

(q) Channel Configurations

In some cases, a device can have a configuration of a device asdescribed in U.S. Pat. No. 8,021,614. In some cases, a channel in adevice comprises mirrored arrays of obstacles, in which one array ofobstacles is configured to deflect a particle of at least apredetermined size to a center bypass channel, and a second array ofobstacles adjacent to the first array of obstacles also directsparticles of at least a first predetermined size to the center bypasschannel. In some cases, the bypass channel comprises a wall thatseparates the first array of obstacles and the second array ofobstacles. In some cases, the bypass channel does not comprise a wallthat separates the first array of obstacles and the second array ofobstacles. In some cases, a channel with a mirrored array comprises atleast two inlets. In some cases, a sample outlet is in fluidcommunication with the bypass channel. In some cases, a channel with amirrored array comprises at least one waste outlet. In some cases, achannel with a mirrored array comprises a plurality of inlets and aplurality or outlets with an array of obstacles disposed betweentherein, wherein each of the arrays in the mirrored array, and whereinthe channel is configured to flow a plurality of fluids from theplurality of inlets to the plurality of outlets. Each of the pluralityof fluids can be flowed parallel to adjacent fluid flows. In some cases,a multi-stream (e.g., ‘car wash’) device comprising a mirrored array asdescribed herein further comprises one or more separator walls betweenadjacent flow streams. In some cases, channel with a mirrored arraycomprises at least one waste outlet.

In some cases, a channel does not comprise a mirrored array. In somecases, a channel comprises a first array of obstacles that directparticles of at least a predetermined size to a bypass channel adjacentto a wall of the channel. In some cases, a channel comprises a pluralityof bypass channels. In some cases, a channel comprises two mirroredarrays of obstacles, two inlets, and a central buffer stream forconcentrating particles from each of the mirrored array of obstacles. Insome cases, a channel comprises one inlet. In some cases, a channelcomprises two inlets.

(r) Flow Properties

In some cases, flow through a device comprising an array of obstacles islaminar.

The methods, compositions, devices, systems, and/or kits describedherein can facilitate rapid flow rate through a device. In some cases, aflow rate through a device is at least 0.01, 0.02, 0.03, 0.04, 0.05,0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1,1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10,10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17,17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105,110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 170, 175, 180,185, 190, 200, 250, 300, 350, 400, 450, or 500 mL/min.

In some cases, a flow rate through a device is about 0.01, 0.02, 0.03,0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8,8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5,16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 170,175, 180, 185, 190, 200, 250, 300, 350, 400, 450, or 500 mL/min.

In some cases, a flow rate through a device is less than 0.01, 0.02,0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5,8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15,15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160,170, 175, 180, 185, 190, 200, 250, 300, 350, 400, 450, or 500 mL/min.

In some cases, a flow rate through a device is about 0.05 to about 0.1mL/min, about 0.1 to about 0.5 mL/min, about 0.5 to about 1 mL/min,about 1 to about 5 mL/min, about 5 to about 10 mL/min, about 10 to about20 mL/min, about 20 to about 50 mL/min, about 50 to about 100 mL/min,about 100 to about 200 mL/min, or about 200 to about 500 mL/min.

In some cases, a fluid velocity is at least 1, 5, 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000,2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 mm/sec.

In some cases, a fluid velocity is about 1, 5, 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000,2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 mm/sec.

In some cases, a fluid velocity is less than 1, 5, 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000,2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 mm/sec.

In some cases, shear stress is about 10, 50, 100, 500, 1000, 5000,10,000, 25,000, 50,000, 75,000, 100,000, 200,000, 300,000, 400,000,500,000, 600,000, 700,000, 800,000, 900,000 or 1,000,000 s⁻¹.

In some cases, shear stress is less than 10, 50, 100, 500, 1000, 5000,10,000, 25,000, 50,000, 75,000, 100,000, 200,000, 300,000, 400,000,500,000, 600,000, 700,000, 800,000, 900,000 or 1,000,000 s⁻¹.

In some cases, shear stress is more than 10, 50, 100, 500, 1000, 5000,10,000, 25,000, 50,000, 75,000, 100,000, 200,000, 300,000, 400,000,500,000, 600,000, 700,000, 800,000, 900,000 or 1,000,000 s⁻¹.

(s) Pressure

In some cases, a sample is flowed through a device at a pressure ofabout 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3,4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 atm.

In some cases, a sample is flowed through a device at a pressure of atleast 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3,4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 atm.

In some cases, a sample is flowed through a device at a pressure of lessthan 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9,3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4,4.5, 4.6, 4.7, 4.8, 4.9, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 atm.

-   -   (t) Predetermined Size (Critical Size)

In some cases, a device as described herein can be used to deflectparticles of at least a predetermined size to a first outlet (e.g.,product) and particles less than a predetermined size to a second (e.g.,waste) outlet. In some cases, a predetermined size is about 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5,5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13,13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 200, 300, 400, 500, 600, 700, 800, 900,or 1000 μm.

In some cases, a predetermined size is less than 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5,7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14,14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 μm.

In some cases, a predetermined size is more than 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5,7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14,14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 μm.

In some cases, a device comprises an array of obstacles, wherein theobstacles comprise obstacles with a diameter of 18 μm, and the array ofobstacles comprise rows or obstacles with a gap of 18 μm, wherein asubsequent row has a 1/42 row shift. In some case, the obstacles aresemi-mirrored. Semi-mirrored can mean that the layout can reflect thearray across the center line (i.e., the collection channel) and thenshift that reflected array down towards the outlet by a certain numberof rows. In some cases, the number of rows is 5. The width of thecollection channel can be kept uniform or substantially more uniformthan if the obstacles were to be truly mirrored. The arrays on eitherside of the collection channel can be identical, but just shifted fromeach other in the direction of the flow.

(u) Obstacle Coating

In some cases, obstacles comprise an affinity capture agent, e.g., anantibody, other protein-binding partner, or nucleic acid. Obstacles cancomprise specific affinity-capture agent to capture specific particlesin a sample. Affinity capture is described, e.g., in PCT Publication No.WO2012094642, which is herein incorporated by reference in its entirety.

(v) On-Chip Cleaning System

In some cases, devices described herein can comprise an integratedsystem for on-chip cleaning. Examples of the self cleaning system areillustrated in FIG. 33A-C. In some cases, the on-chip cleaning systemcomprises openings in walls of a channel such that fluid can be flowedthrough the openings, where the fluid flow is substantiallyperpendicular to the usual flow path of the channel. The cleaning systemcan be used to remove particles, e.g., cells trapped in an array ofobstacles. In some cases, openings are present in only one wall thatbounds a channel. In some cases, openings are present in both walls thatbound a channel.

FIG. 33A illustrates a device comprising a deterministic lateraldisplacement (DLD) array also comprises an on-chip cleaning system.Sample and buffer inputs are illustrated at the left of the channel, andproduct and waste outputs are illustrated at the right. Walls for fluidcontainment are illustrated. The on-chip cleaning system is illustratedin an “open” configuration with openings in the walls that bound the DLDarray. A fluid is illustrated flowing from the top of the schematic tothe bottom of the schematic at a right angle to the particle and bufferflow direction.

FIG. 33B illustrates an on-chip cleaning system in a closedconfiguration. In this configuration, openings in the walls that boundthe channel are blocked.

FIG. 33C illustrates an on-chip cleaning system in an openconfiguration. In this configuration, openings in the walls that boundthe channel are unblocked. In some cases, as illustrated in FIG. 33C,the inlet and outlet ends of the channel can be blocked while theopenings in the wall are in the open position. In some cases, inletand/or outlet ends of the channel are not blocked when the openings inthe wall are in an unblocked configuration.

In some case blocking and unblocking of the openings in the walls iscontrolled manually or automatically. In some cases, blocking andunblocking of the openings in the walls is controlled electronically.

In some cases, an on-chip cleaning system is activated when a sensor istriggered. In some cases, the sensor is a pressure sensor (e.g., ifbackpressure in the device rises above a threshold (e.g., due toclogging), an alert can be sent that the on-chip cleaning system can beutilized, or the on-chip cleaning system can be activatedautomatically). In some case, the sensor is an optical sensor, e.g., anoptical system, e.g., a microscope. In some cases, the optical systemcan monitor the device to detect clogging, e.g., by detection of trappedfluorescent particles. In some cases, the sensor is a spectrophotometerthat detects obstruction of a light path through the bottom and top ofthe device (e.g., reduction in transmission of light through the deviceindicates clogging).

Any device comprising a channel comprising an array of obstacles cancomprise an on-chip cleaning system. The array of obstacles can be asymmetric array of obstacles, asymmetric array of obstacles, mirroredarray of obstacles, a mirrored array of obstacles with a central bypasschannel with or without a wall, or a semi-mirrored array of obstacles.

The on-chip cleaning system can be organized in a variety ofconfigurations. The number of openings in the walls that bound a channelcan be dependent on the length of the channel. In some cases, each wallcomprises a plurality of openings, e.g., at least 2, 5, 10, 20, 50, 75,100, 200, 500, 750, 1000, 5000, or 10,000. In some cases, each wallcomprises at most 2, 5, 10, 20, 50, 75, 100, 200, 500, 750, 1000, 5000,or 10,000 openings. In some cases, each of the openings in a wall is inan unblocked or blocked configuration. In some cases, not all of theopenings in a wall are in an unblocked or blocked configuration. In somecases, at least 2, 5, 10, 20, 50, 70, 90, or 100% of openings in a sideof a wall are configured to be a blocked or unblocked configuration. Insome cases, less than 2, 5, 10, 20, 50, 70, 90, or 100% of openings in aside of a wall are configured to be in a blocked or unblockedconfiguration.

Each of the openings in the walls of a channel can have a diameter of atleast 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 20, 30, 40, or 50 μm. In some cases, each of the openings in awall of channel has a diameter of less than 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 μm.In some cases, each of the openings in a wall of channel has a diameterof about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 30, 40, or 50 μm. In some cases, openings in a wall ofa channel are connected to flow paths. In some cases, each of the flowpaths is under control of the same fluid flow system. In some cases,each of the flow paths is not under control of the same fluid flowsystem.

A flow rate of a cleaning solution using the on-chip cleaning system canbe at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 20, 30, 40, or 50 mL/min.

A Flow rate of a cleaning solution using the on-chip cleaning system canless than 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 30, 40, or 50 mL/min.

A flow rate of a cleaning solution using the on-chip cleaning system canbe about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 30, 40, or 50 mL/min.

Flow through the openings in a wall of a channel can be powered by apump, e.g., syringe pump or high pressure pump. In some cases, operationof an on-chip cleaning system is automated. In some case, operation ofan on-chip cleaning system is conducted through an electronic device,e.g., a computer.

A cleaning solution used in an on-chip cleaning system can comprise oneor more agents, e.g., a detergent (e.g., 2-aminoethyl methanthiosulfonate hydrobromide, CHAPS, CHAPSO, digitonin, lithium dodecylsulfate, n-dodecyl-beta-D-maltopyranoside,n-octyl-beta-d-glucopyranoside, NDSB-195, NDSB-201, NDSB-211, NDSB-221,NDSB-256, NONIDET-P40, Pluronic F68, Pluronic F-127, MTSES, Tween-20,Tween-80, Tween-40, sodium dodecyl sulfate (SDS), Triton X-100, TritonX-114, MTSET, sulfobetaine-10, sulfobetaine-12, or sulfobetaine-14),alcohol (e.g., ethanol, methanol), buffer (e.g., Tris-HCl, Trizma,HEPES, MES, phosphate buffer, potassium buffer), enzyme (DNase, RNase,protease, restriction enzyme), reducing agent (DTT,beta-mercaptoethanol), chelating agent (e.g., EDTA (e.g., 1 mM, 5 mM),EGTA (e.g., 1 mM, 5 mM)), anti-bacterial agent, antibiotic (e.g.,chloramphenicol, ampicillin, kanamycin, erythromycin, gentamicin,neomycin, nelimicin, streptomycin, tobramycin, penicillin, bacitracin,polymyxin B, ciproflaxin, or tetracycline), anti-fungal agent,anti-viral agent, protease inhibitor, acid (e.g., hydrochloric acid,sulfuric acid, tartaric acid, nitric acid, phosphoric acid, boric acid,methanesulfonic acid, acetic acid, citric acid, formic acid, orfluoroacetic acid), base (e.g., NaOH). In some cases, the cleaningsolution comprises about 1 to about 20% ethanol, or about 10 to about20% ethanol, or less than 20% ethanol.

A cleaning solution can comprise F108, which can be a bifunctional blockcopolymer surfactant.

In some cases, multiple solutions are flowed through the cleaning systemin succession.

In some cases, a cleaning solution is applied to a device and is allowedto remain in the device for at least 1, 5, 10, 30, or 60 min, or aboutleast 4, 8, 12, 16, 20, or 24 hrs, or at least 3, 5, 7, 14, 21, or 28days. In some cases, a cleaning solution is applied to a device and isallowed to remain in the device for less than about 1, 5, 10, 30, or 60min, or less than 4, 8, 12, 16, 20, or 24 hrs, or less than 3, 5, 7, 14,21, or 28 days.

In some cases, a cleaning solution is washed out with water.

Example 8 describes use of an on-chip cleaning system.

In some cases, the size-based separation methods described herein do notmake use of a centrifuge and/or sedimentation. In some cases, thesize-based separation methods described herein do make use of acentrifuge or sedimentation.

(w) Taper Angle

An obstacle, or pillar can be cylindrical. In some cases, obstacles on adevice are not perfectly cylindrical. An obstacle, or at least 50% ofobstacles in an array, can have a taper angle of less than 10, 9, 8, 7,6, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.5, 0.4, 0.3, 0.2, or 0.1° Anobstacle can have a taper angle of 0°. An obstacle, or at least 50% ofobstacles in an array, can have a taper angle of about 0.1 to about 1,about 1 to about 2, about 2 to about 3, about 3 to about 4, or about 1to about 4°.

H. Materials of Construction and Surface Chemistry

In some embodiments, a device is made by hot embossing PMMA andpolycarbonate. Due to their low cost and compatibility withreplication-based fabrication methods, thermoplastics can represent anattractive family of materials for the fabrication of lab-on-a-chipplatforms. A diverse range of thermoplastic materials suitable formicrofluidic fabrication is available, offering a wide selection ofmechanical and chemical properties that can be leveraged and furthertailored for specific applications. High-throughput embossing methodssuch as reel-to-reel processing of thermoplastics is an attractivemethod for industrial microfluidic chip production. The use of singlechip hot embossing can be a cost-effective technique for realizinghigh-quality microfluidic devices during the prototyping stage. Methodsfor the replication of microscale features in two thermoplastics,polymethylmethacrylate (PMMA) and/or polycarbonate (PC) are describedherein using hot embossing from a silicon template fabricated by deepreactive-ion etching. Further details can be found in “Microfluidicdevice fabrication by thermoplastic hot-embossing” by Yang and Devoe,Methods Mol. Biol. 2013; 949: 115-23, which is herby incorporated byreference herein in its entirety. In some cases, a device is made ofpolypropylene.

In some cases, a device comprises a polymer. In some cases, a device ismade by injection molding. In some cases, a device is manufactured by aphotolithographic technique. In some cases, a device is manufactured bysoft embossing. In some cases, the embossing occurs on a polymer chip.In some cases, a device comprises plastic.

A device can be sealed and bonded in any suitable manner. The mainchallenge can be bonding planar microfluidic parts together hermeticallywithout affecting the shape and size of micro-sized channels. A numberof bonding techniques such as induction heating are suitable. Thechannels can be fabricated by using Excimer laser equipment. Furtherdetails can be found in “Sealing and bonding techniques forpolymer-based microfluidic devices” by Abdirahman Yussuf, Igor Sbarski,Jason Hayes and Matthew Solomon, which is hereby incorporated byreference herein in its entirety.

Further bonding techniques include Adhesive Bonding, Pressure sensitivetape/Lamination, Thermal Fusion Bonding, Solvent Bonding, Localizedwelding, Surface treatment and combinations thereof. Further details canbe found in “Bonding of thermoplastic polymer microfluidics” by Chia-WenTsao and Don L. DeVoe, Microfluid Nanofluid (2009) 6:1-16, which isherby incorporated by reference herein in its entirety.

In some embodiments, the device is made from a polymer and/or plastic.The polymer and/or plastic can be hydrophilic and/or wettable. Table 4summarizes properties of some plastics.

TABLE 4 Summary of physical properties for common microfluidicthermoplastics Optical CTE Water Solvent Acid/base transmissivityPolymer Acronym T_(g) (° C.) T_(m) (° C.) (10⁻⁶° C.⁻¹) absorption (% )resistance resistance Visible UV^(a) Cyclic olelin (co)polymer COC/COP 70-155 190-320 60-80 0.01 Excellent Good Excellent ExcellentPolymethylmethacrylate PMMA 100-122 250-260  70-150 0.3-0.6 Good GoodExcellent Good Polycarbonate PC 145-148 260-270 60-70 0.12-0.34 GoodGood Excellent Poor Polystyrene PS  92-100 240-260  10-150 0.02-0.15Poor Good Excellent Poor Polypropylene PP −20 160  18-185 0.10 Good GoodGood Fair Polyetheretherketone PEEK 147-158 340-350 47-54 0.1-0.5Excellent Good Poor Poor Polyethylene terephthalate PET 69-78 248-26048-78 0.1-0.3 Excellent Excellent Good Good Polyethylene PE 30 120-130180-230 0.01 Excellent Excellent Fair Fair Polyvinylidene chloride PVDC0  76 190 0.10 Good Good Good Poor Polyvinyl chloride PVC 80 180-210  500.04-0.4  Good Excellent Good Poor Polysulfone PSU 170-187 180-190 55-600.3-0.4 Fair Good Fair Poor T_(m) melting temperature. CTE coefficientof thermal expansion ^(a)high UV transmissivity often requires theselection of special polymer grades, e.g. without stabilizers or otheradditives

A device can be fabricated in any suitable manner. Some techniquesinclude Replica molding, Softlithography with PDMS, Thermoset polyester,Embossing, Injection Molding, Laser Ablation and combinations thereof.Further details can be found in “Disposable microfluidic devices:fabrication, function and application” by Gina S. Fiorini and Daniel T.Chiu, BioTechniques 38:429-446 (March 2005), which is herby incorporatedby reference herein in its entirety. The book “Lab on a Chip Technology”edited by Keith E. Herold and Avraham Rasooly, Caister Academic PressNorfolk UK (2009) is a resource for methods of fabrication, and suchwhich is hereby incorporated by reference herein in its entirety. Adevice can be manufactured by cast molding or reactive injectionmolding.

Exemplary materials for fabricating the devices of the invention includeglass, silicon, steel, nickel, polymers, e.g., poly(methylmethacrylate)(PMMA), polycarbonate, polystyrene, polyethylene, polyolefins, silicones(e.g., poly(dimethylsiloxane)), polypropylene, cis-polyisoprene(rubber), poly(vinyl chloride) (PVC), poly(vinyl acetate) (PVAc),polychloroprene (neoprene), polytetrafluoroethylene (Teflon),poly(vinylidene chloride) (SaranA), and cyclic olefin polymer (COP) andcyclic olefin copolymer (COC), and combinations thereof. Other materialsare known in the art. For example, deep Reactive Ion Etch (DRIE) can beused to fabricate silicon-based devices with small gaps, small obstaclesand large aspect ratios (ratio of obstacle height to lateral dimension).Thermoforming (embossing, injection molding) of plastic devices may alsobe used. Additional methods include photolithography (e.g.,stereolithography or x-ray photolithography), molding, embossing,silicon micromachining, wet or dry chemical etching, milling, diamondcutting, Lithographie Galvanoformung and Abformung (LIGA), andelectroplating. For example, for glass, traditional silicon fabricationtechniques of photolithography followed by wet (KOH) or dry etching(reactive ion etching with fluorine or other reactive gas) may beemployed. Techniques such as laser micromachining can be adopted forplastic materials with high photon absorption efficiency. This techniqueis suitable for lower throughput fabrication because of the serialnature of the process. For mass-produced plastic devices, thermoplasticinjection molding, and compression molding can be suitable. Conventionalthermoplastic injection molding used for mass-fabrication of compactdiscs (which preserves fidelity of features in sub-microns) may also beemployed to fabricate the devices. For example, the device features arereplicated on a glass master by conventional photolithography. The glassmaster is electroformed to yield a tough, thermal shock resistant,thermally conductive, hard mold. This mold serves as the master templatefor injection molding or compression molding the features into a plasticdevice. Depending on the plastic material used to fabricate the devicesand the requirements on optical quality and throughput of the finishedproduct, compression molding or injection molding may be chosen as themethod of manufacture. Compression molding (also called hot embossing orrelief imprinting) can have the advantage of being compatible with highmolecular weight polymers, which can be excellent for small structuresand may replicate high aspect ratio structures but has longer cycletimes. Injection molding can work well for low aspect ratio structuresand can be suitable for low molecular weight polymers. A device can bemade using any of the materials described herein. In some cases, thesurface of the (plastic) device is treated to make it hydrophilic and/orwettable. Surfaces in devices, e.g., microfluidic devices, can play acritical role because they can define properties such as wetting,adsorption and repellency of biomolecules, biomolecular recognitionusing surface-immobilized receptors, sealing and bonding of differentmaterials. In some cases, two types of treatments can be used to modifythe surface properties of a device, e.g., a microfluidics device: wetchemical treatments and gas phase treatments. Wet treatments can besimple in terms of infrastructure requirements; they can be flexible andfast to develop from a research standpoint. Surface treatment of adevice, e.g., microfluidics device, for production can be however bestachieved using dry processes based on plasma and chemical vapordeposition. These treatments can eliminate the need for rinsing anddrying steps, have high throughput capability and are highlyreproducible.

In some cases, the treatment is a wet chemical treatment. Among the wetchemical treatments available, the formation of self-assembledmonolayers (SAMs) is one of the most versatile and easy to use surfacetreatments. SAMs have been developed on metals, silicon oxides andpolymers. Molecules in SAMs can pack closely and can be composed of aheadgroup that can bind covalently to the substrate, an alkyl chain anda terminal functional group. The thickness of the SAM can depend on thelength of the alkyl chain and density of the molecules on the surfaceand is typically a few nanometers. SAMs can be easy to prepare and canbe patterned with sub-micrometer lateral resolution. Different terminalgroups can be used for defining the wetting properties of the surface aswell as the affinity for or repellency of proteins. For glass surfaces,oxides and polymers that can be oxidized, grafting alkylsiloxanes tosurfaces might be the simplest and most economical method. A wettabilitygradient from superhydrophobic to hydrophilic can be achieved bysuperposing a SAM-based wetting gradient onto microstructures in siliconthat have varying lateral spacing.

Polymeric SAMs can comprise block copolymers and can have variousthree-dimensional structures, which can give the opportunity to varytheir mode of grafting to a surface and the types of functionalitiesthat they carry. Such layers can reach a significant thickness ofseveral hundreds of nanometers and protect/functionalize surfaces morereliably than thinner monolayers. For example, apoly(oligo(ethyleneglycol)methacrylate) polymer brush can coat glassdevices, e.g., chips, e.g., microfluidic chips to make them hydrophilicand antifouling.

Coating polymers onto surfaces to modify their properties is possible.For example, poly(ethyleneglycol) can be used to “biologically”passivate device, e.g., microfluidic device materials and can be graftedonto PMMA surfaces of capillary electrophoresis microchips to make themhydrophilic. Poly(tetrafluoroethylene) can be used to make chemicallyresistant devices, e.g., microfluidic devices. Polymeric materialsemployed to fabricate devices, e.g., microfluidic devices, can bemodified in many ways. In some cases, functional groups such as aminesor carboxylic acids that are either in the native polymer or added bymeans of wet chemistry or plasma treatment are used to crosslinkproteins and nucleic acids. DNA can be attached to COC and PMMAsubstrates using surface amine groups. Surfactants such as Pluronic® canbe used to make surfaces hydrophilic and protein repellant by addingPluronic® to PDMS formulations. In some cases, a layer of PMMA is spincoated on a device, e.g., microfluidic chip and PMMA is “doped” withhydroxypropyl cellulose to vary its contact angle.

Proteins can be used on surfaces to change surface wettability, topassivate a surface from non-specific protein binding and forfunctionalization. Proteins readily adsorb to hydrophobic substratessuch as PDMS and polystyrene. By exploiting this property, PDMSsubstrates can be coated with neutravidin to immobilize biotinylatedproteins or biotinylated dextran. Antibody coatings can be optimizeddepending on the hydrophobicity of the polymeric substrate. Bovine serumalbumin can be used protein to passivate surfaces from non-specificadsorption and is easy to deposit spontaneously from solution tohydrophobic surfaces. On a hydrophilic substrate, a layer of hydrophobicpoly(tetrafluoroethylene) can first be coated to enable the subsequentdeposition of bovine serum albumin. Heparin, a biological moleculewidely used as an anticoagulant, can be deposited from solution ontoPDMS to make channels, e.g., microchannels hydrophilic while preventingadhesion of blood cells and proteins.

In some embodiments, a device undergoes a gas phase treatment. Plasmaprocessing not only can modify the chemistry of a polymeric surface butit also can affect its roughness significantly thereby exacerbatingwetting properties to make surfaces superhydrophilic and fluorocarbonscan be plasma deposited to make surfaces superhydrophobic. Polymericsurfaces can be patterned using ultraviolet light to initiate radicalpolymerization followed by covalent grafting of polymers. Plasma-inducedgrafting can be used to attach poly(ethyleneglycol) onto polyamide andpolyester surfaces to render them antifouling. Dextran can be apolysaccharide comprising many glucose molecules that can be coated tomake hydrophilic antifouling surfaces. In some cases, a starting pointto modifying polymers is to introduce surface hydroxyl groups using aplasma treatment followed by grafting a silane and dextran layer.Similarly, PDMS can be superficially oxidized using ultraviolet lightfor grafting a dextran hydrogel.

The large surface to volume ratio of devices, e.g., microfluidicstructures can make any potential surface-analyte/reagent interaction apotential issue. Therefore, irrespective of the method used to treat thesurfaces of a microfluidic device for POC testing, in some cases thesurfaces of the device can not attract and deplete analytes orbiochemicals that are needed for the test. In some cases, surfacetreatments do not interfere with signal generation and acquisitionprinciples of the device. Further details can be found in “Capillarymicrofluidic chips for point of care testing: from research tools todecentralized medical diagnostics” a thesis by Luc Gervais, Ecolepolytechnique federale de Lausanne, 23 Jun. 2011, which is herbyincorporated by reference herein in its entirety.

To reduce non-specific adsorption of cells or compounds, e.g., releasedby lysed cells or found in biological samples, onto the channel walls,one or more channel walls may be chemically modified to be non-adherentor repulsive. The walls may be coated with a thin film coating (e.g., amonolayer) of commercial non-stick reagents, such as those used to formhydrogels. Additional examples chemical species that may be used tomodify the channel walls include oligoethylene glycols, fluorinatedpolymers, organosilanes, thiols, poly-ethylene glycol, hyaluronic acid,bovine serum albumin, poly-vinyl alcohol, mucin, poly-HEMA,methacrylated PEG, and agarose. Charged polymers may also be employed torepel oppositely charged species. The type of chemical species used forrepulsion and the method of attachment to the channel walls can dependon the nature of the species being repelled and the nature of the wallsand the species being attached. Such surface modification techniques arewell known in the art. The walls may be functionalized before or afterthe device is assembled. The channel walls may also be coated in orderto capture materials in the sample, e.g., membrane fragments orproteins.

V. Properties of Particles Flowed Through Devices

The methods, compositions, devices, systems, and/or kits describedherein can be used for high-throughput processing (e.g., chemical and/orenzymatic treatment), purification, isolation, and/or concentration ofparticles. The methods, compositions, devices, systems, and/or kitsdescribed herein can be used to isolate particles with relatively highpurity, yield, and/or viability if the particles are living, e.g., cellsor organisms). One or more samples can be applied to one ore more inletson a device. One or more buffers can be applied to one or more inlets ona device. Particles of at least a critical (predetermined) size can bepassed through an array of obstacles and be deflected to one outlet, andparticles less than the critical size can pass to another outlet.

An array of obstacles can comprise any cross-sectional shape, obstaclediameter, gap size, tilt angle, and/or array pattern geometry describedherein.

Temperature of a flowing liquid, or ambient temperature, or temperatureof a device, can be about −20, −10, 0, 4, 10, 15, 20, 22, 23, 24, 25,26, 30, 35, 37, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100°C. Temperature of a flowing liquid, or ambient temperature, ortemperature of a device, can be less than −20, −10, 0, 4, 10, 15, 20,22, 23, 24, 25, 26, 30, 35, 37, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, or 100° C. Temperature of a flowing liquid, or ambienttemperature, or temperature of a device, can be more than −20, −10, 0,4, 10, 15, 20, 22, 23, 24, 25, 26, 30, 35, 37, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, or 100° C. Temperature of a flowing liquid, orambient temperature, or temperature of a device can be about −20 toabout −10° C., about −10 to about 0° C., about 0 to about 4° C., about 4to about 25° C., about 25 to about 30° C., about 30 to about 37° C.,about 37 to about 50° C., about 50 to about 65° C., or about 65 to about100° C.

A. Purity

In some cases, methods, compositions, devices, systems, and/or kitsdescribed herein can be used to isolate first particles, e.g., cellsthat are about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure.

In some cases, methods, compositions, devices, systems, and/or kitsdescribed herein can be used to isolate first particles, e.g., cellsthat are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure.

In some cases, methods, compositions, devices, systems, and/or kitsdescribed herein can be used to isolate first particles, e.g., cells,that are about 1 to about 10% pure, about 10% to about 20% pure, about20% to about 30% pure, about 30% to about 40% pure, about 40% to about50% pure, about 50% to about 60% pure, about 60% to about 70% pure,about 70% to about 80% pure, about 80% to about 90% pure, or about 90%to about 100% pure.

In some cases, devices and methods described herein are used to isolateleukocytes from whole blood. In some cases, devices and methodsdescribed herein remove over 99% of erythrocytes, platelets, plasmaproteins, and unbound staining from leukocytes. In some cases,leukocytes are removed from serum

B. Yield

In some cases, methods, compositions, devices, systems, and/or kitsdescribed herein can be used to give a yield of about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, or 100% of first particles, e.g., cells from a sample.

In some cases, methods, compositions, devices, systems, and/or kitsdescribed herein can be used to give a yield of at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, or 100% of first particles e.g., cells from a sample.

In some cases, methods, compositions, devices, systems, and/or kitsdescribed herein can be used to give a yield of about 1% to about 10%,about 10% to about 20%, about 20% to about 30%, about 30% to about 40%,about 40% to about 50%, about 50% to about 60%, about 60% to about 70%,about 70% to about 80%, about 80% to about 90%, or about 90% to about100% of first particles, e.g., cells from a sample.

In some cases, devices and methods described herein are used to isolateleukocytes from whole blood. In some cases, at least 80%, 85%, or 90% ofleukocytes are recovered from a whole blood sample without introducingbias among the leukocyte population.

C. Viability

In some cases, particles in a sample are alive (e.g., cell or organism).In some cases, methods, compositions, devices, systems, and/or kitsdescribed herein can be used to isolate particles (e.g., cells,organisms) that are about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% viable.

In some cases, methods, compositions, devices, systems, and/or kitsdescribed herein can be used to isolate particles (e.g., cells,organisms) that are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% viable.

In some cases, methods, compositions, devices, systems, and/or kitsdescribed herein can be used to isolate particles (e.g., cells,organisms) that are at about 1% to about 10% viable, about 10% to about20% viable, about 20% to about 30% viable, about 30% to about 40%viable, about 40% to about 50% viable, about 50% to about 60% viable,about 60% to about 70% viable, about 70% to about 80% viable, about 80%to about 90% viable, or about 90% to about 100% viable.

In some cases, a sample comprises leukocytes and erythrocytes. In somecases, the method, compositions, devices, systems, and/or kits describedherein can be used to process (e.g., chemical and/or enzymatictreatment), wash, and then isolate leukocytes from a sample such thatthe leukocytes are greater than 90% pure (i.e. less than 10%erythrocytes), greater than 90% of the leukocytes in the sample areisolated (greater than 90% yield), and greater than 90% of theleukocytes in the sample are viable.

VI. Applications

These devices and methods described herein can be a general replacementfor centrifugation in cell processing. Leading suppliers of clinical andresearch instruments can be searching for alternatives to the currentcell processing methods (3). In some cases, the methods using a DLDmicrofluidic technology are as described in U.S. Patent Publication No.US2010/0059414.

In some cases, a device as provided herein is used for Bead-basedimmune- and nucleic-acid assays (e.g., Luminex assays, BD CBA).

In some cases, a device as provided herein is used for cell-surfaceimmunofluorescence.

In some cases, a device as provided herein is used for cell surfacelabeling (e.g., immunofluorescence) combined with intra-cellularlabeling.

In some cases, a device as provided herein is used for Leukemiaphenotyping and genotyping including washing away interfering solubleblood components

In some cases, a device as provided herein is used for in-situ nucleicacid analysis (e.g., fluorescence in-situ hybridization).

In some cases, a device as provided herein is used for cell cyclesynchronization by size selection of cell cycle phases.

In some cases, a device as provided herein is used to purify by size,label, and concentrate cells from small cell samples like cerebrospinalfluid.

In some cases, a device as provided herein is used for industrialfunctionalized particle manufacturing (e.g. antibody labelledmicro-particles for bioassays).

In some cases, a device as provided herein is used to remove largeparticles/cells for sensitive detection of bacteria, virus, or exosomesin biological samples (e.g., blood).

In some cases, up to at least 8 centrifugal wash/concentrate steps canbe reduced to 3 on-chip processes of <5-10 min each for 0.1-1 ml samples(e.g., blood) (FIG. 16). Downstream applications can go far beyond flowcytometry, and can range from laboratory research to existing and newclinical diagnostics.

A. DLD Microfluidic Technology to Wash and Concentrate Leukocytes fromBlood.

As shown in FIG. 16, conventional processes for labeling of leukocytes(left) and the reduction of the up to 8 centrifugal wash/concentratesteps to 3 on-chip microfluidic steps (middle and right) using a deviceas provided herein. The embodiment going vertically down the centerreplaces each centrifugal wash/concentrate step with an on-chipwash/concentrate process. The embodiment going vertically down the rightavoids erythrocyte lysis entirely,isolating/harvesting/washing/concentrating leukocytes (and leukemiacells) from blood samples in a single step after the Surface Labelingstep.

DLD separation can outperform standard centrifugal procedures. Forexample, processing (e.g., on-chip microfluidic Washing/Concentrating) asample comprising leukocytes and labeling reagent (e.g., antibodies) ina device as provided herein, can harvest >90% of leukocytes at >90%viability while removing >99% unbound fluorescent or Fix/Perm reagentswith no skewing of sub-populations.

FIG. 17A shows a DLD array designed to “bump” E. coli (>1 um size). Thebacterial suspension is input on the left, and flows left to right,confined by the walls of the microfluidic device. The micropost arraycauses the fluorescent (GFP-containing) bacteria (seen as the whiteblurred band) to flow along the tilted array axis, so that they movedown to accumulate against the lower array wall, while the fluid streamcontinues straight ahead. The bacteria concentrate along the lower edgeof the device, seen as the growing bold white streak. By separatelycollecting this concentrated output from its own output channel, distantfrom the waste fluid output channel, bacteria can be concentrated by50-fold during the ˜100 secs they took to flow across the DLD device.

The device shown in FIG. 17A can be extended to include 2 input streams:stream 1 can be a suspension of leukocytes after labeling with Mabs (ortreatment with Fix/Perm reagents); stream 2 can be buffer fluid (asshown in FIG. 17B). The redesigned bump array can cause large cells,such as leukocytes (>8 um diameter; and leukemia cells, ˜8-20 um) tomove at an angle to the input fluid flow, whereas dissolved molecules orsmall suspended particles or molecules in solution (e.g. immunomagneticbeads, fluorescent Mabs, Fix/Perm reagents) tend to move left to right,in or following the fluid flow. The microchip can operate at lowReynolds number, so the flow is laminar and not turbulent; thus, the 2streams move in parallel. As in FIG. 17A, the desired cells (leukocytesand leukemia cells) concentrate at the lower edge of the array. Byrouting most of the output fluid to a waste channel and the concentratedcells to a product channel, washing and concentration can be achieved atthe same time.

In addition to leukocyte harvesting, DLD technology can achieveconcurrent washing of the cells. Whole blood can be incubated with CD45FITC for 30 minutes at room temperature, then leukocytes can be removedfrom the blood using a device as provided herein. A reduction influorescence in the cell-free product of ≧99% can be achieved afterleukocyte harvesting on the device, indicating efficient removal offluorescent Mab.

As shown in FIG. 17: (a) Top view: Example of DLD mechanism showinguniform input of fluorescent (white) E. coli bacteria in a tilted postarray being bumped downwards at an angle to the fluid flow, to becomehighly concentrated against the lower array wall and then collected,while fluid moves horizontally (7). (b) Schematic “washing” ofleukocytes and/or leukemia cells by extension of FIG. 17A by adding aninput buffer stream. Only large leukocytes can move down to the bufferstream and lower wall. (c) Time-lapse image of leukocytes (blue fromnuclear stain) being harvested out of a stream of blood (reddish/white)moving left to right using a chip with design as in FIG. 17B. Note thatthe tilted array of microposts can be seen.

In some cases, 0.1-1 ml of blood are processed in <5-10 minutes, with aleukocyte yield >90%, no skewing of cell types compared to input cellsor conventional methods, and >99% removal of unbound fluorescent Mab andFix/Perm reagents. In some cases, the cells are moved away from theinput stream faster than the input stream widens due to diffusion towardthe clean buffer stream. While diffusion coefficients of the largeparticles, e.g., leukocytes and leukemia cellscan be negligible to firstorder approximation, the diffusion coefficients of the Mabs or Fix/Permreagents are generally not negligible. In some cases, these dissolved orsuspended molecules are much smaller than cells and thus have a highdiffusion coefficient. Unwanted spreading of these reagents can causecontamination of the leukocyte output. Increasing the tilt angle canhelp prevent spreading, but can reduce the gap between the posts for afixed cell size, which can be undesirable due to occasional very largecells. Another option is to lengthen the chip, since the celldisplacement can be linear with length of the array and the spreading(diffusion) increases only as the square root. However, this can havethe drawback of requiring a more expensive chip. In some embodiments,DLD is microscopically a deterministic process, not a random one, suchas gel electrophoresis. Thus, running a DLD microfluidic process fastercannot change the path of the desired cells, and high speed can reducethe time for unwanted reagent diffusion. In some cases, the fluid speedis ˜0.1 mm/sec. Thus, running through a chip of typical length (˜3 cm)can take 5 minutes, which can be too slow not only for the goal ofleukocyte throughput, but also to prevent the unwanted diffusion. Insome cases, the bumping process operates well with little or minimalcelldamage even at speeds of >100 mm/sec (i.e. flow rates >1 ml/min) Flowspeed can be varied as required to reduce reagent contamination of theoutput. Qualitative images of results with E. coli (5) indicate thatthis diffusion problem for washing away reagents can be overcome atmodest flow speeds.

A second potential challenge is the wide range of cell size of differenttypes of leukocytes and leukemia cells. This can be addressed by usingtriangular instead of round posts, which allows for a larger gap betweenposts than with round posts, due to flow anisotropies in the gap.Finally, after Fix/Perm, the cells can be “stiffer” than before, andthus act as if they have a different diameter in the DLD chip. If thisis observed, a DLD chip with a slightly larger critical size can beneeded for cells after Fix/Perm.

B. Microfluidic Leukocyte Harvesting and DLD Wash/Concentrate in aSingle Step

The conventional erythrocyte Lysis Step and the subsequent centrifugalWash/Concentrate Step can be replaced by a single DLD microfluidic step.In addition to >90% yield and viability with thorough removal oflabeling and Fix/Perm reagents, some embodiments also achieve thismicrofluidic Wash/Concentrate system to deplete >99% of erythrocytesfrom a whole blood sample incubated with fluorescent Mabs.

Because they are larger size than red blood cells, leukocytes can bebumped out from an input stream of whole or diluted blood in a DLD chip.In some cases, a device as provided herein is configured to directlysurface label leukocytes present in blood (without any lysis or removalof erythrocytes), followed by harvesting, washing, and concentrating theimmunostained leukocytes directly from the blood (FIG. 17C). This canallow the complete leukocyte preparation process to be accomplished withonly 3 on-chip Wash/Concentrate steps. Note that the microchip can bedesigned so that the smaller red blood cells (from unlysed blood),platelets, and non-cellular plasma constituents are not bumped; thus,the output can contain only the harvested, washed, and concentratedleukocytes.

In some cases, an input stream comprises leukocytes in whole blood withMabs added thereto. The blood can be diluted with running buffer. Insome cases, a larger volume of input is required due to the inputcomprising concentrated leukocytes, larger amounts of Mabs (tocompensate for the dilution factor) are thereby required for optimalimmunostaining.

In some instances, cells are immunostained exactly as described herein,except the starting cell preparation can comprise unlysed blood, ratherthan lysed blood. Immunostained cells can undergo the developed on-chipLeukocyte Harvest/Wash/Concentrate Step, then enumerated by flowcytometry. Results can be compared vs. cells immunostained after aconventional erythrocyte Lysis Step. Statistical comparisons ofviability, yield, purity, and leukocyte subsets can be performed.

In some embodiments, 99% of erythrocytes are removed (i.e. obtainleukocytes <10% contaminated by erythrocytes). In some cases, theviscosity of blood (due to the 1000-fold higher numbers of erythrocytes)is higher compared to a suspension of leukocytes in buffer. This canchange the internal dynamics of the flow patterns near the boundarybetween the buffer and the blood. At least three approaches can be usedto solve this problem: (a) driving the blood input and the buffer inputat different pressures, (b) replacing the pressure-driven approach witha fixed flow rate (syringe pump) approach, and (c) diluting the blood(e.g. 3-5-fold) to reduce its viscosity. The latter approach can be themost straightforward, although it can require higher flow rates toachieve throughput targets. In some cases, an output is achieved thatconcentrates leukocytes by 30-fold from the (diluted) input. Inpractice, this can require a fairly wide (and thus long) chip, which canbe limited by the ˜100 mm starting wafer size. Options include using afabrication facility capable of larger wafer sizes (e.g. 200 mm), orcascading chips—one chip does the harvesting and initial concentration(FIG. 17C), and then the fluid flows through a second chip designed forconcentration (like that of FIG. 17A). Finally, if the triangular postapproach for wide gaps does not eliminate clogging due to anomalouslylarge cells (e.g. >30 um), pre-filtering may be performed, or a thirdchip in series can be used to remove >30 um-sized cells.

In some embodiments, a device as provided herein replaces conventionallysis and centrifugal steps for harvesting leukocytes (and leukemiacells) from blood, and centrifugal Wash/Concentrate steps after cellsurface labeling, Fix/Perm, and intracellular labeling with rapid andrepeatable on-chip processes, as described herein.

In some cases, the method resembles a “Car Wash” approach, in which(analogously) a car is subjected to multiple sequential treatments (e.g.wash, rinse, wax, dry) as it moves through the car wash process (FIG.18A). Building on the concept of FIG. 18B, blood enters a chip, and thedesired cells are moved through sequential parallel streams of chemicals(labels, fix/perm reagents, etc.) to accomplish one step after another.In some cases, blood which has already been labeled with a cell surfacemarker (but not washed) enters a device as provided herein in the topstream (flowing left to right), and relatively large leukocytes andleukemia cells are induced to flow downwards at an angle to the fluidflow by the DLD bumping process to be harvested out of the blood. Thecells then flow through a stream for fixing and permeabilizing thecells' membranes, then through a stream for intracellular labeling.Finally, the cell surface/intracellular labeled cells are washed andconcentrated and collected at the bottom edge of the array.

On-chip labeling of cells by moving them into a labeling stream andsubsequent removal of the labeled cells from the labeling stream can bedone using previously isolated but unlabeled blood platelets as theinput and a CD41 fluorescent label for the labeling stream (FIG. 18B).On-chip lysis of cells by moving them across a stream of lysis agentscan be performed. On-chip lysis is not required for the “Car Wash” ofFIG. 18A, but it provides the possibility of on-chip sequential chemicalprocessing for steps such as Fix/Perm prior to intracellular staining.Required incubation times and concentrations, yields, and broadening ofthe incubation or Fix/Perm streams due to diffusion can be determined.

FIG. 18A shows a schematic view of a “Car Wash” concept for multiplesequential chemical treatment processing on chip for a cell preparation.A single continuous-flow process combines all steps in FIG. 16 into asingle chip. FIG. 18B shows false-color fluorescent time-lapse image ofplatelets moving downwards in a DLD array across 3 parallel on-chipstreams for on-chip label and wash. Upper Stream: input of unlabeled(invisible) platelets; Middle stream: phycoerythrin-conjugated CD41label; Lower Stream: labeled platelets in wash buffer.

C. NGS Library Generation Using DLD Array

In some cases, the devices, methods, compositions, systems and/or kitsprovided herein are used for processing (e.g., chemical and/or enzymaticprocessing or treating) a sample from a cell to a nucleic acid. Theprocessing can be serial. In some cases, the devices, methods,compositions, systems and/or kits provided herein are used for seriallyprocessing a sample from a plurality of cells to a plurality of nucleicacids, wherein the plurality of nucleic acids comprise nucleic acids ina nucleic acid library. Devices, methods, and/or systems are described,e.g., in PCT Publication No. WO2013020089, which is herein incorporatedby reference in its entirety. The cell can be processed (e.g., chemicaland/or enzymatic processing or treating) using a device herein to highmolecular weight (“HMW”) nucleic acid using at least one chemical and/orenzymatic reagent stream flowing through at least one DLD bump array.The nucleic acid library can be configured for use in a sequencingplatform. The sequencing platform can be any next generation sequencingplatform known in the art. In some cases, a device as provided herein toprocess (e.g., chemical and/or enzymatic processing or treating) cellsto nucleic acid is used to process a high volume of a sample comprisingthe cells. The sample can be at least 10 ml. In some cases, a device asprovided herein to process (e.g., chemical and/or enzymatic processingor treating) cells to nucleic acid is used to process (e.g., chemicaland/or enzymatic processing or treating) at a high flow rate. In somecases, the devices as provided herein to process (e.g., chemical and/orenzymatic processing or treating) cells in a sample comprising cellsfrom cells to nucleic acids comprises at least one separator wall asprovided herein. The at least one separator wall is configured toseparate a flow stream comprising a reagent (e.g., chemical and/orenzymatic reagent as provided herein) from an adjacent flow stream. Insome cases, the devices as provided herein to process (e.g., chemicaland/or enzymatic processing or treating) cells in a sample comprisingcells from cells to nucleic acids comprises an ‘on-chip’ self cleaningsystem as provided herein. In some cases, the devices as provided hereinto process (e.g., chemical and/or enzymatic processing or treating)cells in a sample comprising cells from cells to nucleic acids comprisesat least one separator wall, and an ‘on-chip’ self cleaning system asprovided herein.

In some cases, a HMW nucleic acid as isolated by devices and methodsprovided herein has an effective hydrodynamic radius that is greaterthan a critical size of the DLD array in a device provided herein. Themethod can include receiving the HMW nucleic acid at at least one bumparray, and contacting the HMW nucleic acid with at least one chemicaland/or enzymatic reagent stream, wherein the at least one chemicaland/or enzymatic reagent stream flows in the direction of bulk fluidflow through the bump array, whereas the HMW nucleic acid flows at anangle to the direction of bulk fluid flow. The HMW nucleic acid canreact with the at least one chemical and/or enzymatic reagent stream.

In some cases, a device and/or system as provided herein includes atleast one bump array (e.g., DLD array) device that has one or more bumparrays. The bump array device can serially treat and purify nucleic acidfluid samples. Multiple cycles of treatment and purification can becarried out using a single flow device in a single continuous flowoperation. The treatments can be chemical and/or enzymatic. The nucleicacids can be purified from cells and/or complex liquid biologicalsample, such as whole blood. The bump array device can also be used forperforming various processing of the purified nucleic acids.Non-limiting examples of such processing can include at least one of thefollowing: phosphorylation, dephosphorylation, restriction digestion,ligation, denaturation, hybridization, processing by polymerases,fluorescent or radioactive labeling, chemical modification of DNA basesor backbone groups, enzymatic or chemical excision of modified bases,staining of nucleic acids with chromophores or fluorophores, etc. and/orothers and/or any combination thereof. The nucleic acids can be particlebound nucleic acids, where nucleic acids can be attached tomicroparticles. This can allow for processing of small nucleic acids.The particles can render the attached nucleic acids bumpable in arrayswith easily manufactured array dimensions.

In some cases, a device and/or system as provided herein is used in amethod for processing of fluids. The processing can include purificationof fluids which can be accomplished by flowing a complex fluid sampleinto a bump array, using a bump array to isolate nucleic acid containingcells or particles of interest on the basis of particle size, using abump array to contact isolated particles with one or one reagent streamsthat can release nucleic acid from the particles in substantially pureform, and using a bump array to move purified nucleic acids out of thereagent stream. Once the purified nucleic acids are moved out of thereagent stream, the purified nucleic acids can be substantially freefrom other cellular and sample components and can be substantially freefrom reagent stream components of the bump array.

In some cases, a device and/or system as provided herein comprises aseries of bump (e.g., DLD) arrays connected in series so that theproduct output of one bump array is connected to a sample input of asubsequent individual bump array. In some cases, a single bump array isused for all steps. Cell fractionation and reagent treatments can beaccomplished in physically distinct regions of a single bump array. Theinput sample can be avian or mammalian blood and thenucleic-acid-containing particles can be white blood cells. The inputsample can be avian or mammalian blood and the nucleic-acid-containingparticles can be circulating tumor cells. The input sample can be avianor mammalian whole blood and the nucleic-acid-containing particles canbe white blood cells, bacteria, viruses, fungi, parasitic protozoansand/or any others as provided herein and/or any combination thereof.

In some cases, a device and/or system as provided herein provide aserial processing of high molecular weight nucleic acids by chemicaland/or enzymatic means on devices as provided herein comprising bump(e.g., DLD) arrays. The HMW nucleic acid can have an effectivehydrodynamic diameter that can be greater than the critical diameter ofthe bump array and the HMW nucleic acid can be contacted with at least afirst reagent stream, where the first reagent stream can flow in thedirection of bulk fluid flow and where the HMW nucleic acid is bumpedthrough the first reagent stream and can react with the first reagents.

In some cases, a device and/or system as provided herein provide aserial processing of HMW nucleic acids by one or more chemical orenzymatic means that can be accomplished by flowing a sample of HMWnucleic acids into a bump array, using a bump array to contact HMWnucleic acids with at least one reagent streams that can modify thenucleic acids (e.g., chemically, enzymatically, etc.), and, optionally,using a bump array to remove purified nucleic acids from the reagentstream.

In some cases, a device and/or system as provided herein comprisesindividual bump arrays connected in a series of bump arrays so that theproduct output of one bump array is connected to the sample input of asubsequent individual bump array in the series. The bump arrays can bethe same bump arrays (and the cell fractionation and reagent treatmentscan be accomplished in physically distinct regions of one continuousbump array). In some cases, a DNA sample is capable of being bound(covalently or noncovalently) to microparticles, wherein themicroparticles are bumpable, whereby the microparticles act as carriersto take the DNA through modification reactions in subsequent reagentstreams.

In some cases, a device and/or system as provided herein provides forprocessing of whole blood to produce a pure nucleic acid. A deviceand/or system as provided herein can be used to produce a modified purenucleic acid. The modified pure nucleic acid can be a DNA sequencinglibrary and/or a recombinant DNA library.

In some cases, a device as provided herein is used in a system that canaccept a whole blood sample as input and produce a genomic DNA librarysuitable for next-generation sequencing (“NGS”). Library constructioncan take place in a single automated process without any userintervention. The system can lower the cost and labor of NGS sequencingand accelerate movement of NGS technology into diagnostic settings. Thesystem can be scalable to accommodate samples containing very few cells(e.g., a single cell level), which can be important in treatment ofcancer and/or other important medical problems or large sample (e.g., atleast 10 ml), which can be important for high-throughput techniques.

In some cases, a device and/or system as provided herein includes amicrofluidic, continuous-flow design. Liquid samples containingparticles (e.g., cells, nuclei, and large macromolecules such asrandomly-coiled HMW DNA) can be pumped through flow cells that can bepopulated by a regular array of micron-sized posts. The spacing andalignment of the posts can be arranged so that particles above a certaincritical size can be “bumped” by the posts into a flow path that runsdiagonally across the direction of bulk liquid flow. In contrast, samplecomponents smaller than the critical size can travel straight along withthe bulk flow. Using this mechanism, larger sample components can beseparated and purified from smaller components laterally across thechip.

Samples can flow through these bump arrays under conditions of laminarflow (Reynolds number, R_(e), <<1), so that discrete reagent streams canbe introduced into arrays without significant lateral mixing. Largeparticles can be bumped diagonally into, and out of, such reagentstreams to perform chemical or enzymatic reactions on the particles. Thedevices as provided herein can be used to purify leukocyte nuclei,purify DNA, and enzymatically modify DNA for generation of NGSlibraries.

In some cases, a device and/or system as provided herein is used in amethod for sequentially processing blood samples using bump arrays. Afew microliters (“μl”) of blood can be obtained. Blood cells can beseparated from plasma. Cells can be washed with a buffer stream as theyare separated from the plasma. In some cases, the cells are lysed.Washed cells can be lysed by bumping them through a reagent streamcontaining non-ionic detergent. The non-ionic detergent can be anynon-ionic detergent known in the art. After removing the lysis reagent,intact leukocyte nuclei can be bumped diagonally through a wash bufferstream. Cytoplasmic contents too small to bump (e.g., below a criticalsize of a DLD bump array) can be carried out of the array in thedetergent lysis stream. Chromosomal DNA can be isolated and/or purified.Washed leukocyte nuclei can be bumped through a nuclear lysis reagentstream to remove all lipid and nuclear proteins from HMW chromosomalDNA. The nuclear lysis reagent stream can comprise a protein denaturant(e.g., guanidine isothiocyanate) for extracting nucleic acid (e.g. HWchromosomal DNA) from proteins (e.g., histone proteins). In some cases,guanidine isothiocyanate is present in a separate stream from thenuclear lysis stream. Array dimensions in a device as provided hereincan be chosen so that HMW DNA, in its double-stranded random-coilconfiguration, can be bumped diagonally out of the lysis reagent stream.All or substantially all nuclear lipids, RNA, and proteins too small tobump (e.g., below a critical size of a DLD bump array) can be carriedout of the array in the lysis stream. In some cases purified DNA isreacted with a transposase-adapted reagent to generate librarycointegrates. Purified HMW DNA can be bumped through a reagent streamcontaining a transposase complex that can be preloaded withsequencing-adapter-modified transposon ends. A method using a device asprovided herein can provide a transposase complex in which thetransposon-adapted ends of the transposasome can be on the same linearpiece of DNA. As a result, a reaction of the transposasome with the HMWDNA can generate a colinear insertion product that can increase the sizeof the HMW DNA target. The target DNA can remain bumpable, wherein thetarget DNA can be separated from the transposasome reagent stream andunreacted adapter DNA. HMW cointegrates can be purified from thetransposase reagent stream. Cointegrates can be reacted with restrictionenzyme to generate a sequencing library. The library can be separatedfrom uncut HMW DNA and recovered from the bump array. In some cases, afinal sequencing library produced by a method using a device and/orsystem as provided herein can be cleaved from the HMW co-integrate DNAby bumping the DNA through a restriction enzyme reagent stream. Theenzyme can cleave engineered sites in the modified transposon that liejust outside of the sequencing adapters. The cleaved library can be lowin molecular weight (about 200-2000 bp), and can be no longer bumpable.The library can be removed from the array in the restriction enzymestream. Uncleaved, unreacted HMW DNA can be bumped out of the reagentstream diagonally (and can be recovered).

In some cases, the devices, methods, compositions, systems and/or kitsprovided herein include micron-sized post arrays with high structuralrigidity and high aspect ratios for the purposes of processing fluidsamples. The bump arrays can be manufactured from silicon, cyclic olefinresin, molded plastic disposable flow cells, as well as any othermaterials.

In some cases, the devices, methods, compositions, systems and/or kitsprovided herein separate or fractionate, analyze, and/or collectpurified or processed polynucleic acid analytes or fractions derivedfrom a raw biological sample as provided herein.

In some cases, the devices, methods, compositions, systems and/or kitsprovided herein process smaller nucleic acid molecules by attaching thenucleic acids to microparticles that are adapted to be bumped in a bump(e.g., DLD array). The microparticles can act as carriers fortransporting the attached nucleic acids through reagent streams formodification of the nucleic acids. For example, emulsion PCR withprimer-modified microparticles can be used for generation of DNAsequencing template beads (Ion Torrent and 454 sequencing methods;Rothberg et al. 2011. Nature v475, pp 348-352; Margulies et al., Nature.V437, pp 376-380). In some cases, emulsion PCR methods can be used forevaluation of the frequency of rare mutant genes in tissue from cancerpatients (Vogelstein's “BEAMing” method, Diehl et al. Nature Methods.2006 v3 pp 551-559). In some cases, the devices, methods, compositions,systems and/or kits provided herein are used to process particle-basedemulsion PCR by combining washing, denaturation, and primerhybridization into a single bump array process. For example, a bumparray can be designed with post spacing chosen so that the criticaldiameter of the array can be less than that of the microparticles usedfor the emulsion PCR. This can ensure that the microparticles can bebumped consistently at all positions within the array. After emulsionPCR, the emulsion can be broken and the aqueous particles fraction canbe fed into a device comprising a bump array as provided herein near theupper left hand corner. As particles enter the array, the particles canbe deflected toward an opposing boundary wall, while the PCR reagentscan flow downward in the direction of bulk flow. A suitable wash buffercan be fed into the top of the array immediately to the right of theparticle input port. As the particles are bumped out of the inputstream, the particles can pass through the wash buffer stream, which canclean away additional PCR reagent. As the particles move further downthe array, the particles can enter a denaturing reagent stream (e.g.,which can contain about 20-200 mM KOH or NaOH with about 1-10 mM EDTA),which can convert the double-stranded amplicons on the particles tosingle-stranded form. The non-covalently bound amplicon strand can bewashed down the array with the denaturant stream, and the particles canbe bumped rightward into a neutralizing buffer that can be suitable forhybridization reactions in the next step. Generally, such neutralizingbuffer can contain a buffer (for example, 20-200 mM Tris-HCl, pH7.5-8.0) and monovalent ions to support hybridization (for example,20-500 mM NaCl). The particles can then be bumped through ahybridization reagent stream containing sequencing primer (in the caseof the 454/Ion Torrent applications), or labeled oligonucleotide probe(in the case of the BEAMing application). The reagent stream can haveoligo probes in the low micromolar concentration range (about 0.1micromolar to about 50 micromolar), and can have about the same ionicstrength as the neutralizing buffer stream described above. The ionicstrength can be adjusted higher or lower as needed to achieve thecorrect stringency of hybridization. In the final processing step of thearray, the hybridized particles can be bumped out of the hybridizationstream into a final wash buffer stream. This final wash buffer is chosenaccording to the downstream application to be used (sequencing in thecase of 454/Ion applications, fluorescent particle sorting in the caseof the BEAMing assay). The hybridized, washed particles are collectedfrom the output port of the final wash buffer located near the lowerright corner of the array.

VII. Downstream Applications

Particles processed (e.g., chemically or enzymatically processed ortreated), purified, isolated, and/or concentrated using methods,compositions, devices, systems, and/or kits described herein can bestored and/or used in downstream applications. Described herein arevarious applications for particles that have been processed (e.g.,chemically processed or treated), purified, isolated, and/orconcentrated using methods, compositions, devices, systems, and/or kitsdescribed herein

Although this disclosure discusses leukocyte and stem cell processingfor flow cytometry the same technology can be used for multiple existingand new cellular and other (e.g. DNA, RNA) tests for cancer and otherdiseases.

In some cases, the devices, compositions, systems, kits and/or methodsdescribed herein are used to prepare samples for nucleic acid (e.g., DNAor RNA) sequencing. Nucleic acids can be isolated from any type of cellincluding prokaryotic, eukaryotic, archaea, single celled organisms,multi-cellular organisms or tissues (e.g., plants or animals), and thelike. The nucleic acid can be sequenced in any manner, including singlemolecule or shotgun sequencing, in a nanopore, by detecting a change inpH upon nucleotide incorporation events, by fluorescence detection ofincorporated or released dyes, etc. . . . . The cells are lysed andnucleic acid is sorted from cellular debris using the post arrays asdescribed herein. The nucleic acid can be concentrated to any suitableconcentration and/or purified to any suitable purity (e.g., at least70%, at least 80%, at least 90%, at least 95%, at least 99%, at least99.9%, and the like).

A. Blood Banking (e.g., Cryopreservation)

In some cases, blood is separated into components using devices andmethods described herein, and the components are stored. In some cases,erythrocytes are isolated or purified. In some cases, erythrocytes arestored at from about 1 to about 6° C. In some cases, erythrocytes arestored for about 20 to about 60 days, or about 30 to about 50 days, orup to 42 days. In some cases, erythrocytes are frozen (e.g., with acryoprotectant, e.g., glycerol) and stored at, e.g., less than −60° C.,e.g., for at least 10 years. In some cases, stored erythrocytes are usedfor transfusion. In some cases, isolated erythrocytes are administeredto a patient after trauma, surgery, blood loss, or a patient with ablood disorder, e.g., sickle cell anemia.

In some cases, plasma is isolated and frozen for later use. In somecases, plasma is stored for up to a year. Plasma can be administered toa subject, e.g., a burn patient, subject in shock, or subject with ableeding disorder.

In some cases, platelets are isolated. In some cases, platelets areisolated, e.g., for transfusion. In some cases, isolated platelets arestored at room temperature, e.g., for about 5 to 7 days. In some cases,platelets are administered to a subject with cancer, an organtransplant, or a subject who is undergoing, or has undergone, surgery.

In some cases, an isolated blood component is cryoprecipitatedanti-haemophilic factor (cryoprecipitated AHF). Cryoprecipitated AHF canbe stored frozen for about a year. In some cases, Cryoprecipitated AHFis administered to a subject with hemophilia or Von Willebrand disease.

Other blood components that can be isolated and stored are granulocytes.In some cases, granulocytes are used transfusion within 24 hrs aftercollection. In some cases, granulocytes are administered to subject totreat infections that are unresponsive to antibiotic therapy.

In some cases, lymphocytes are isolated and may be stored, with orwithout gene modification, prior to administration to patients withcancer or infectious or other diseases.

In some cases, purified particles, e.g., cells, e.g., stem cells, e.g.,HSCs are preserved, e.g., cryopreserved. Methods of cryopreservation aredescribed, e.g., in Berz et al (2007) Cryopreservation of HematopoieticStem Cells. Am J Hematol. 82: 463-472, which is herein incorporated byreference. In some cases, a heparinized plasmalyte solution and/ordimethylsulfoxide (DMSO) (e.g., 10% DMSO) are added to purifiedparticles, e.g., cells, e.g., HSCs. In some cases, the purifiedparticles, e.g., HSCs are in a solution with a final concentration ofDMSO of less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7,6, 5, 4, 3, 2, 1, or 0.5% DMSO. In some cases, particles, e.g., cells,e.g., HSCs, are in a solution with a final DMSO concentration of about 2to about 10%, or about 5% to about 15%. In some cases, leukocytes arecryopreserved.

In some cases, purified particles, e.g., cells, e.g., stem cells, e.g.,HSCs are combined with saline and or serum albumin. In some cases, acryoprotectant is hydroxyethyl starch (HES), propylene glycol, alphatocopherol, catalase, ascorbic acid, trehalose, or capase inhibitor(e.g., zVAD-fmk). In some cases, a cryoprotectant is a glycol (e.g.,ethylene glycol, propylene glycol, or glycerol). In some cases, acryoprotectant is 2-Methyl-2,4-pentanediol (MPD). In some cases, acryoprotectant is sucrose. In some cases, purified particles, e.g.,cells, e.g., stem cells, e.g., HSCs are mixed with more than onecryoprotectant.

In some cases, purified particles, e.g., cells, e.g., stem cells, HSCsare frozen to a temperature of less 5° C., than −79° C., less than −155°C. or less than −195° C. In some cases, particles, e.g., cells, e.g.,stem cells, e.g., HSCs are frozen to a temperature of about 4° C. ofabout −80° C., −156° C. or −196° C. In some cases, purified particles,e.g., cells, e.g., stem cells, e.g., HSCs are frozen to from about −196°C. to about −80° C. In some cases, purified particles, e.g., cells,e.g., stem cells, e.g., HSCs are stored in a liquid phase of a nitrogentank. In some cases, purified particles, e.g., cells, e.g., stem cells,e.g., HSCs are store in a vapor nitrogen phase.

In some cases, purified particles, e.g., cells, e.g., stem cells, e.g.,HSCs are frozen at a controlled rate freezing, e.g., at a rate of 1-2°C./min up to a temperature point of about −40° C. Then, the freezingprocess down to a target of −120° C. is performed can be performed afaster pace, about 3-5° C./min. In some cases, purified particles, e.g.,cells, e.g., HSCs are cooled to a temperature of −4° C., then placed ina freezer at −80° C.

In some cases, purified particles, e.g., cells, e.g., stem cells, e.g.,HSCs are cryopreserved for at least 1, 10, 30, 180, or 365 days. In somecases, HSCs are cryopreserved for at least 1, 5, 10, 20, 30, 50, 75, or100 years.

In some cases, purified particles, e.g., cells, e.g., stem cells, e.g.,HSCs are cryopreserved at a density of less than 10⁻¹¹, 10⁻¹⁰, 10⁻⁹,10⁻⁸, 10⁻⁷, 10⁻⁶, 10⁻⁵ cells/mL. In some cases, purified particles,e.g., cells, e.g., stem cells, e.g., HSCs are cryopreserved at a densityof at least 10⁻¹¹, 10⁻¹⁰, 10⁻⁹, 10⁻⁸, 10⁻⁷, 10⁻⁶, 10⁻⁵ cells/mL.

In some cases, cryopreserved purified particles, e.g., cells, e.g., stemcells, e.g., HSCs are thawed at 37° C. (e.g., in a water bath, gelpads). In some cases, cyropreserved purified particles, e.g., cells,e.g., HSCs are thawed at a temperature of about 0° C. to about 37° C.

In some cases, a cryopresevative (e.g., DMSO) is washed out of purifiedparticles, e.g., cells, e.g., HSC sample after thawing. In some cases, athawed purified particle, e.g., cell, e.g., stem cell, e.g., HSC sampleis diluted in human serum albumin (HSA) (e.g., 2.5%) and dextran 40(e.g., 5%). The sample can then be centrifuged or passed through amicrofluidic device described herein, e.g., at a temperature of 10° C.In some cases, an HSA/dextran solution is added to the purifiedparticles, e.g., cells, e.g., stem cells, e.g., HSCs again. In somecases, the DMSO concentration is less than 1.7%, e.g., washing and/ordilution. In some cases, stem cells, e.g., HSCs with a DMSOconcentration of less than 1.7% is infused in a subject.

In some case, cyropreserved purified particles, e.g., cells, e.g., stemcells, e.g., HSCs are stored in a container. In some cases, a containeris an ethinyl vinyl acetate (EVA) container. In some cases, a containeris gamma irradiated. In some cases, a container is a stainless steelcontainer. In some cases, a container comprises, PVC, polyolefin, orpolyethelene. In some cases, a container comprises Teflon, Kaplon, FEP,and/or polyimide.

In some cases, purified cells, e.g., stem cells, e.g., HSCs areevaluated by cell counting for total nucleated cells and CD34+ cells(e.g., by flow cytometry); trypan blue exclusion for viability,7-acinoactinomycin for viability, or propidium iodide for viability;engraftment in NOD/SCID (immunodeficient mice), or a clonogenic assay(e.g., CFU-Sd12 assay in mice; CFU-GM; CFU-GEMM; BFU-E, or LTC-IC).

B. Cancer Treatment

In some cases, purified, isolated, and/or concentrated stems cells,e.g., HSCs can be used to treat cancer, e.g. cancer of the blood, e.g.,leukemia or lymphoma. In some cases, purified, isolated, and/orconcentrated stem cells, e.g., HSCs are obtained from a subject andsubsequently administered to the same subject. The stem cells can travelto the bone marrow and begin to produce new blood cells.

In some cases, stem cells, e.g., HSCs are obtained from a first subjectand administered to second subject (e.g., relative, e.g., sister orbrother of the first subject). In some cases, the first subject andsecond subject are not relatives. In some cases, the second subject is amatched donor. In some cases, the first subject and the second subjecthave similar human leukocyte antigens. In some cases, the first subjectand the second subject do not have similar human leukocyte antigens. Insome cases, a subject diagnosed with or suspected of having, acutelymphoblastic leukemia, acute myeloblastic leukemia, chronicmyelogoneous leukemia (CML), Hodgkin's disease, multiple myeloma, ornon-Hodgkin′ lymphoma is administered HSCs.

In some cases, administration of stem cells to a subject comprises useof an intravenous (IV) line. In some cases, the transplant takes about 1to about 5 hours. After entering the blood stream, the cells can travelto the bone marrow. Engraftment (normal blood production) can occurwithin about 2 to about 4 weeks after transplantation. In some cases,the methods, compositions, devices, systems, and kits described hereinare used to monitor engraftment.

In some cases, a subject receives a bone marrow transplant (BMT). Insome cases, a subject receives a peripheral blood stem cell transplant(PBSCT). In some cases, a transplant is an autologous transplant (thesubject receive his/her own stem cells).

In some cases, a transplant is a syngeneic transplant (a subjectreceives stem cells from his/her identical twin). In some cases, atransplant is an allogeneic transplant (a subject receives stem cellsfrom his/her brother, sister, parent, or person unrelated to thesubject.

In some cases, stem cells are purified from bone marrow in the pelvicbone or sternum.

In some cases, PBSCs are processed and/or purified by apheresis orleukapheresis. In some cases, stem cells are processed and/or purifiedfrom umbilical cord or placenta

In some cases, processed, purified, isolated, or concentrated stemcells, e.g., HSCs are administered to a subject with CML, and thesubject is also administered imatinib mesylate (Gleevec™). In somecases, the subject is administered stem cells, e.g., HSCs withoutreceiving imatinib mesylate.

In some cases, a subject who receives stem cells e.g., HSCs is resistantto chemotherapy.

In some cases, a subject who receives stem cells, e.g., HSCs is anewborn, infant, child, teenager, young adult, middle aged person, orelderly person.

In some cases, subject who receives stem cells, e.g., HSCs hasneuroblastoma, Ewing's sarcoma, desmoplatic small-round cell tumor, orchronic granulomatous disease.

In some cases, a mini-transplant is used. In some cases, a tandemtransplant is used, involving two sequential courses of high-dosechemotherapy and stem cell transplant.

In some cases, a subject, e.g., a cancer patient, is administeredradiation or chemotherapy, and the radiation or chemotherapy targetshematopoietic cells, which can be destroyed by radiation orchemotherapy. In some cases, processed, purified, isolated, and/orconcentrated HSCs from the subject can be transplanted into the subjectto replace cells destroyed by chemotherapy. Introducing the subject'sown HSCs can reduce the chance of immune mismatch or graft-versus-hostdisease. In some cases, only CD34+, Thy-1+ cells are transplanted intothe subject.

In some cases, stem cells are administered to a subject who is inremission (signs and symptoms of cancer have disappeared).

In some cases, the transplantation of stem cells processed and/orpurified using methods and devices described herein can result in areduction of the risk of graft versus host disease by a least 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, or 100% relative to transplantation of stem cellspurified by conventional methods.

In some cases, a subject who is administered stem cells has one or moreof the following cancers: acute myeloid leukemia; bladder cancer,including upper tract tumors and urothelial carcinoma of the prostate;bone cancer, including chondrosarcoma, Ewing's sarcoma, andosteosarcoma; breast cancer, including noninvasive, invasive, phyllodestumor, Paget's disease, and breast cancer during pregnancy; centralnervous system cancers, adult low-grade infiltrative supratentorialastrocytoma/oligodendroglioma, adult intracranial ependymoma, anaplasticastrocytoma/anaplastic oligodendroglioma/glioblastoma multiforme,limited (1-3) metastatic lesions, multiple (>3) metastatic lesions,carcinomatous lymphomatous meningitis, nonimmunosuppressed primary CNSlymphoma, and metastatic spine tumors; cervical cancer; chronicmyelogenous leukemia (CML); colon cancer, rectal cancer, anal carcinoma;esophageal cancer; gastric (stomach) cancer; head and neck cancers,including ethmoid sinus tumors, maxillary sinus tumors, salivary glandtumors, cancer of the lip, cancer of the oral cavity, cancer of theoropharynx, cancer of the hypopharynx, occult primary, cancer of theglottic larynx, cancer of the supraglottic larynx, cancer of thenasopharynx, and advanced head and neck cancer; hepatobiliary cancers,including hepatocellular carcinoma, gallbladder cancer, intrahepaticcholangiocarcinoma, and extrahepatic cholangiocarcinoma; Hodgkindisease/lymphoma; kidney cancer; melanoma; multiple myeloma, systemiclight chain amyloidosis, Waldenstrom's macroglobulinemia;myelodysplastic syndromes; neuroendocrine tumors, including multipleendocrine neoplasia, type 1, multiple endocrine neoplasia, type 2,carcinoid tumors, islet cell tumors, pheochromocytoma, poorlydifferentiated/small cell/atypical lung carcinoids; Non-Hodgkin'sLymphomas, including chronic lymphocytic leukemia/small lymphocyticlymphoma, follicular lymphoma, marginal zone lymphoma, mantle celllymphoma, diffuse large B-Cell lymphoma, Burkitt's lymphoma,lymphoblastic lymphoma, AIDS-Related B-Cell lymphoma, peripheral T-Celllymphoma, and mycosis fungoides/Sezary Syndrome; non-melanoma skincancers, including basal and squamous cell skin cancers,dermatofibrosarcoma protuberans, Merkel cell carcinoma; non-small celllung cancer (NSCLC), including thymic malignancies; occult primary;ovarian cancer, including epithelial ovarian cancer, borderlineepithelial ovarian cancer (Low Malignant Potential), and less commonovarian histologies; pancreatic adenocarcinoma; prostate cancer; smallcell lung cancer and lung neuroendocrine tumors; soft tissue sarcoma,including soft-tissue extremity, retroperitoneal, intra-abdominalsarcoma, and desmoid; testicular cancer; thymic malignancies, includingthyroid carcinoma, nodule evaluation, papillary carcinoma, follicularcarcinoma, Hiirthle cell neoplasm, medullary carcinoma, and anaplasticcarcinoma; uterine neoplasms, including endometrial cancer or uterinesarcoma.

In some cases, stem cells, e.g., HSCs are processed, purified, isolated,and/or concentrated, e.g., from an HLA-matched subject, and the HSCs aretransplanted into another person, e.g., a sibling of the subject,wherein the sibling has cancer. In some cases, the transplanted HSCsshow antitumor activity (graft-verus-tumor treatment of cancer).

In some cases, Natural Killer (NK) cells are used in immunotherapy,e.g., for cancer, e.g., leukemia. Uses of NK cells are described, e.g.,in Grywacz et al. (2008) Use of natural killer cells as immunotherapyfor leukaemia. Best Pract Res Clin Haematol. 3: 467-483 and Miller(2013) Therapeutic applications: natural killer cells in the clinic.Hematology 2013:247-253, which are herein incorporated by reference intheir entireties.

C. Cancer Diagnosis

In some cases, cells isolated using the methods, compositions, devices,systems, and kits described herein are used to diagnose a cancerdescribed herein, e.g., a blood cancer, e.g., leukemia, lymphoma, ormyeloma. In some cases, the leukemia is adult acute lymphoblasticleukemia, childhood acute lymphoblastic leukemia, adult acute myeloidleukemia, childhood acute myeloid leukemia, chronic lymphocyticleukemia, chronic myelogenous leukemia, or hairy cell leukemia. In somecases, the lymphoma is AIDS-related lymphoma, cutaneous T-cell lymphoma,adult Hodgkin Lymphoma, childhood Hodgkin Lymphoma, mycosis fungoides,adult Non-Hodgkin Lymphoma, childhood Non-Hodgkin Lymphoma, primaryCentral Nervous System Lymphoma, Sézary Syndrome, cutaneous T-CellLymphoma, or Waldenström Macroglobulinemia. In some cases, the bloodcancer is a chronic myeloproliferative disorder, Langerhans cellhistiocytosis, multiple myeloma, plasma cell neoplasm, a myelodysplasticsyndrome, a myelodysplastic/myeloproflierative neoplasm.

In some cases, leukocytes are evaluated with a leukemia and lymphomaresearch panel. In some cases, a research panel is used to look for setsof proteins, e.g., cell surface and/or intracellular proteins that serveas markers for subtypes of normal leukocytes and hematologicmalignancies. In some cases, the panel is evaluated with flow cytometry.The panel can be, e.g., a BD Euroflow multicolor antibody panel (seehttp://www.bdbiosciences.com/eu/documents/EuroFlow_datasheet_new.pdf).The marker in the BD Euroflow multicolor antibody panel can be, e.g.,CD-11c CD22, CD24, CD45, CD49d, CD 123, Igk, CD10, CD27, CD38, CD43,CD81, TCRγδ, β-2 microglobulin, CD9, CD71, CD79b, Igλ, IREM-2 (CD300e),CD2, CD3, CD4, CD7, CD8, CD16, CD16, CD20, CD23, CD36, CD38, CD41a,CD42a, CD45, CD56, CD64, CD105, CD138, CD200, Igλ, Igκ and, HLA-DR. Thelabel (e.g., fluorochrome) associated with the marker in the BD Euroflowmulticolor antibody panel can be FITC, PE, V450, PE-Cy™7, PerCP-Cy5.5,APC-H7, V500-C, APC, PacB, or PacO.

In some cases, leukocytes recovered using devices and/or methodsdescribed herein are evaluated in B-cell analysis (kappa and lambdaratio). Comparing the ratio of kappa-to-lambda can be used to determinewhether a subject might have a plasma cell tumor, e.g., multiplemyeloma, monoclonal gammopathy of undetermined significance (MGUS),Smoldering myeloma, solitary plasmacytoma of the bone, or ALamyloidosis.

In some cases, free light chain production is assessed, which can beprognostic of a worse outcome in multiple myeloma or chronic lymphocyticleukemia.

D. Blood Disorders

In some cases, processed (e.g., chemically and/or enzymaticallyprocessed or treated), purified, isolated, or concentrated HSCs areadministered to a subject with a hereditary blood disorder. Thehereditary blood disorder can be, e.g., aplastic anemia,beta-thalassemia, Blackfan-Diamond syndrome, globoid cellleukodystrophy, sickle-cell anemia, severe combined immunodeficiency,X-linked lymphoproliferative syndrome, or Wiskott-Aldrich syndrome.Inborn errors of metabolism that can be treated with bone marrowtransplants include: Hunter's syndrome, Hurler's syndrome, Lesch Nyhansyndrome, and osteopetrosis. In some cases, the hereditary blooddisorder is Fanconi anemia.

In some cases, processed (e.g., chemically processed or treated),purified, isolated, or concentrated HSCs are administered to a subjectto treat a blood disorder, e.g., amyloidois, anemia, essentialthrombocythemia, Fanconi anemia, Gaucher disease, hemochromatosis,hemolytic anemia, hemophilia, hypereosinophilia, idiopathicthrombocytopenic purpura, an inherited bone marrow failure syndrome,iron-deficiency anemia, Langerhan Cell histiocytosis, leucopenia,mastocytosis, myelofibrosis, a myeloprofilerative disorder, perniciousanemia, polycythermia vera, porphyria, sickle cell anemia, athalassemia, thrombocytopenia, thrombocytosis, thromboticthrombocytopenic purpura, or von Willebrand disease.

In some cases, particles (e.g., cells) processed (e.g., chemicallyand/or enzymatically processed or treated), purified, isolated, and/orconcentrated using methods described herein are used to diagnose a blooddisorder, e.g., an blood disorder described herein.

E. Autoimmune Disease

Stem cells, e.g., HSCs processed (e.g., chemically and/or enzymaticallyprocessed or treated), purified, isolated, and/or concentrated using themethods, compositions, devices, systems, and/or kits described hereincan be administered to a subject to treat an autoimmune disease. In somecases, stem cells, e.g., HSCs purified, isolated, and/or concentratedusing the methods, compositions, devices, systems, and/or kits can beadministered to a subject with an autoimmune disorder that affectsheart, brain, nerves, muscle, skin, eye, joint, lung, kidney, gland, thedigestive tract, or blood vessels. In some cases, an autoimmune disordercan be rheumatoid arthritis, Graves' disease, thryrioditis, scleroderma,systemic sclerosis, vitiligo, systemic lupus erythematosus (SLE),alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis,dermatomyositis, diabetes (type 1), juvenile idiopathic arthritis,glomerulonephritis, Guillain-Barré syndrome, idiopathic thrombocytopenicpurpura, myasthenia gravis, myocarditis, multiple sclerosis,pemphigus/pemphigoid, pernicious anemia, polyarteritis nodosa,polymyositis, primary biliary cirrhosis, psoriasis, scleroderma/systemicsclerosis, Sjögren's syndrome, uveitis, or granulomatosis withpolyangiitis (Wegener's).

F. Other Uses of Stem Cells

In some cases, stem cells processed (e.g., chemically and/orenzymatically processed or treated), purified, isolated, and/orconcentrated using the methods, compositions, devices, systems, and/orkits described herein can be used to treat Alzheimer's diseases, spinalcord injury, stroke, burns, heart disease, or osteoarthritis. In somecases, stem cells can be used to treat cardiovascular disease.

In some cases, stem cells are used to treat a subject with aneurological or neurocognitive condition. The neurological orneurocognitive condition can be a neurological disorder listed on theNational Institute of Neurological Disorders and Stroke webpage(www.ninds.nih.gov/disorders/disorder_index.htm). In some embodiments,the subject can have a sign or symptom. The neurological orneurocognitive condition, or symptom, can be, e.g., abarognosis (e.g.,loss of the ability to detect the weight of an object held in the handor to discern the difference in weight between two objects), acid lipasedisease, acid maltase deficiency, acquired epileptiform aphasia, absenceof the septum pellucidum, acute disseminated encephalomyelitis, adie'spupil, Adie's syndrome, adrenoleukodystrophy, agenesis of the corpuscallosum, agnosia, Aicardi syndrome, Aicardi-Goutieres syndromedisorder, AIDS—neurological complications, akathisia, alcohol relateddisorders, Alexander disease, Alien hand syndrome (anarchic hand),allochiria, Alpers' disease, altitude sickness, alternating hemiplegia,Alzheimer's disease, amyotrophic lateral sclerosis, anencephaly,aneurysm, Angelman syndrome, angiomatosis, anoxia, Antiphospholipidsyndrome, aphasia, apraxia, arachnoid cysts, arachnoiditis,arnold-chiari malformation, Asperger syndrome, arteriovenousmalformation, ataxia, ataxias and cerebellar or spinocerebellardegeneration, ataxia telangiectasia, atrial fibrillation, stroke,attention deficit hyperactivity disorder, auditory processing disorder,autism, autonomic dysfunction, back pain, Barth syndrome, Battendisease, becker's myotonia, Behcet's disease, bell's palsy, benignessential blepharospasm, benign focal amyotrophy, benign intracranialhypertension, Bernhardt-Roth syndrome, bilateral frontoparietalpolymicrogyria, Binswanger's disease, blepharospasm, Bloch-Sulzbergersyndrome, brachial plexus birth injuries, brachial plexus injury,Bradbury-Eggleston syndrome, brain or spinal tumor, brain abscess, brainaneurysm, brain damage, brain injury, brain tumor, Brown-Sequardsyndrome, bulbospinal muscular atrophy, CADASIL (cerebral autosomaldominant arteriopathy subcortical infarcts and leukoencephalopathy),Canavan disease, Carpal tunnel syndrome, causalgia, cavernomas,cavernous angioma, cavernous malformation, Central cervical cordSyndrome, Central cord syndrome, Central pain syndrome, central pontinemyelinolysis, centronuclear myopathy, cephalic disorder, ceramidasedeficiency, cerebellar degeneration, cerebellar hypoplasia, cerebralaneurysm, cerebral arteriosclerosis, cerebral atrophy, cerebralberiberi, cerebral cavernous malformation, cerebral gigantism, cerebralhypoxia, cerebral palsy, cerebral vasculitis,Cerebro-Oculo-Facio-Skeletal syndrome (COFS), cervical spinal stenosis,Charcot-Marie-Tooth disease, chiari malformation, Cholesterol esterstorage disease, chorea, choreoacanthocytosis, Chronic fatigue syndrome,chronic inflammatory demyelinating polyneuropathy (CIDP), chronicorthostatic intolerance, chronic pain, Cockayne syndrome type II,Coffin-Lowry syndrome, colpocephaly, coma, Complex regional painsyndrome, compression neuropathy, concussion, congenital facialdiplegia, congenital myasthenia, congenital myopathy, congenitalvascular cavernous malformations, corticobasal degeneration, cranialarteritis, craniosynostosis, cree encephalitis, Creutzfeldt-Jakobdisease, cumulative trauma disorders, Cushing's syndrome, Cytomegalicinclusion body disease (CIBD), cytomegalovirus infection, Dancingeyes-dancing feet syndrome (opsoclonus myoclonus syndrome), Dandy-Walkersyndrome (DWS), Dawson disease, decompression sickness, De morsier'ssyndrome, dejerine-klumpke palsy, Dejerine-Sottas disease, Delayed sleepphase syndrome, dementia, dementia-multi-infarct, dementia-semantic,dementia-subcortical, dementia with lewy bodies, dentate cerebellarataxia, dentatorubral atrophy, depression, dermatomyositis,developmental dyspraxia, Devic's syndrome, diabetes, diabeticneuropathy, diffuse sclerosis, Dravet syndrome, dysautonomia,dyscalculia, dysgraphia, dyslexia, dysphagia, dyspraxia, dyssynergiacerebellaris myoclonica, dyssynergia cerebellaris progressiva, dystonia,dystonias, Early infantile epileptic, Empty sella syndrome,encephalitis, encephalitis lethargica, encephalocele, encephalopathy,encephalopathy (familial infantile), encephalotrigeminal angiomatosis,encopresis, epilepsy, epileptic hemiplegia, erb's palsy, erb-duchenneand dejerine-klumpke palsies, erythromelalgia, essential tremor,extrapontine myelinolysis, Fabry's disease, Fahr's syndrome, fainting,familial dysautonomia, familial hemangioma, familial idiopathic basalganglia calcification, familial periodic paralyses, familial spasticparalysis, Farber's disease, febrile seizures, fibromuscular dysplasia,fibromyalgia, Fisher syndrome, floppy infant syndrome, foot drop,Foville's syndrome, friedreich's ataxia, frontotemporal dementia,Gaucher's disease, generalized gangliosidoses, Gerstmann's syndrome,Gerstmann-Straussler-Scheinker disease, giant axonal neuropathy, giantcell arteritis, Giant cell inclusion disease, globoid cellleukodystrophy, glossopharyngeal neuralgia, Glycogen storage disease,gray matter heterotopia, Guillain-Barre syndrome, Hallervorden-Spatzdisease, head injury, headache, hemicrania continua, hemifacial spasm,hemiplegia alterans, hereditary neuropathies, hereditary spasticparaplegia, heredopathia atactica polyneuritiformis, herpes zoster,herpes zoster oticus, Hirayama syndrome, Holmes-Adie syndrome,holoprosencephaly, HTLV-1 associated myelopathy, HIV infection, Hughessyndrome, Huntington's disease, hydranencephaly, hydrocephalus,hydrocephalus-normal pressure, hydromyelia, hypercortisolism,hypersomnia, hypertension, hypertonia, hypotonia, hypoxia,immune-mediated encephalomyelitis, inclusion body myositis,incontinentia pigmenti, infantile hypotonia, infantile neuroaxonaldystrophy, Infantile phytanic acid storage disease, Infantile refsumdisease, infantile spasms, inflammatory myopathy, inflammatorymyopathies, iniencephaly, intestinal lipodystrophy, intracranial cyst,intracranial hypertension, Isaac's syndrome, Joubert syndrome, Karaksyndrome, Kearns-Sayre syndrome, Kennedy disease, Kinsbourne syndrome,Kleine-Levin syndrome, Klippel feil syndrome, Klippel-Trenaunay syndrome(KTS), Kluver-Bucy syndrome, Korsakoff s amnesic syndrome, Krabbedisease, Kugelberg-Welander disease, kuru, Lafora disease, lambert-eatonmyasthenic syndrome, Landau-Kleffner syndrome, lateral femoral cutaneousnerve entrapment, Lateral medullary (wallenberg) syndrome, learningdisabilities, Leigh's disease, Lennox-Gastaut syndrome, Lesch-Nyhansyndrome, leukodystrophy, Levine-Critchley syndrome, lewy body dementia,Lipid storage diseases, lipoid proteinosis, lissencephaly, Locked-Insyndrome, Lou Gehrig's, lumbar disc disease, lumbar spinal stenosis,lupus-neurological sequelae, lyme disease-neurological sequelae,Machado-Joseph disease (spinocerebellar ataxia type 3), macrencephaly,macropsia, megalencephaly, Melkersson-Rosenthal syndrome, Menieresdisease, meningitis, meningitis and encephalitis, Menkes disease,meralgia paresthetica, metachromatic leukodystrophy, metabolicdisorders, microcephaly, micropsia, migraine, Miller fisher syndrome,mini-stroke (transient ischemic attack), misophonia, mitochondrialmyopathy, Mobius syndrome, Moebius syndrome, monomelic amyotrophy, mooddisorder, Motor neurone disease, motor skills disorder, Moyamoyadisease, mucolipidoses, mucopolysaccharidoses, multi-infarct dementia,multifocal motor neuropathy, multiple sclerosis, multiple systematrophy, multiple system atrophy with orthostatic hypotension, musculardystrophy, myalgic encephalomyelitis, myasthenia-congenital, myastheniagravis, myelinoclastic diffuse sclerosis, myoclonic encephalopathy ofinfants, myoclonus, myopathy, myopathy-congenital, myopathy-thyrotoxic,myotonia, myotonia congenita, myotubular myopathy, narcolepsy,neuroacanthocytosis, neurodegeneration with brain iron accumulation,neurofibromatosis, Neuroleptic malignant syndrome, neurologicalcomplications of AIDS, neurological complications of lyme disease,neurological consequences of cytomegalovirus infection, neurologicalmanifestations of AIDS, neurological manifestations of pompe disease,neurological sequelae of lupus, neuromyelitis optica, neuromyotonia,neuronal ceroid lipofuscinosis, neuronal migration disorders,neuropathy-hereditary, neurosarcoidosis, neurosyphilis, neurotoxicity,neurotoxic insult, nevus cavernosus, Niemann-pick disease, Non 24-hoursleep-wake syndrome, nonverbal learning disorder, normal pressurehydrocephalus, O'Sullivan-McLeod syndrome, occipital neuralgia, occultspinal dysraphism sequence, Ohtahara syndrome, olivopontocerebellaratrophy, opsoclonus myoclonus, Opsoclonus myoclonus syndrome, opticneuritis, orthostatic hypotension, Overuse syndrome, chronic pain,palinopsia, panic disorder, pantothenate kinase-associatedneurodegeneration, paramyotonia congenita, Paraneoplastic diseases,paresthesia, Parkinson's disease, paroxysmal attacks, paroxysmalchoreoathetosis, paroxysmal hemicrania, Parry-Romberg syndrome,Pelizaeus-Merzbacher disease, Pena shokeir II syndrome, perineuralcysts, periodic paralyses, peripheral neuropathy, periventricularleukomalacia, persistent vegetative state, pervasive developmentaldisorders, photic sneeze reflex, Phytanic acid storage disease, Pick'sdisease, pinched nerve, Piriformis syndrome, pituitary tumors, PMG,polio, polymicrogyria, polymyositis, Pompe disease, porencephaly,Post-polio syndrome, postherpetic neuralgia (PHN), postinfectiousencephalomyelitis, postural hypotension, Postural orthostatictachycardia syndrome, Postural tachycardia syndrome, Prader-Willisyndrome, primary dentatum atrophy, primary lateral sclerosis, primaryprogressive aphasia, Prion diseases, progressive hemifacial atrophy,progressive locomotor ataxia, progressive multifocalleukoencephalopathy, progressive sclerosing pohodystrophy, progressivesupranuclear palsy, prosopagnosia, Pseudo-Torch syndrome,Pseudotoxoplasmosis syndrome, pseudotumor cerebri, Rabies, Ramsay huntsyndrome type I, Ramsay hunt syndrome type II, Ramsay hunt syndrome typeIII, Rasmussen's encephalitis, Reflex neurovascular dystrophy, Reflexsympathetic dystrophy syndrome, Refsum disease, Refsumdisease-infantile, repetitive motion disorders, repetitive stressinjury, Restless legs syndrome, retrovirus-associated myelopathy, Rettsyndrome, Reye's syndrome, rheumatic encephalitis, rhythmic movementdisorder, Riley-Day syndrome, Romberg syndrome, sacral nerve root cysts,saint vitus dance, Salivary gland disease, Sandhoff disease, Schilder'sdisease, schizencephaly, schizophrenia, Seitelberger disease, seizuredisorder, semantic dementia, sensory integration dysfunction,septo-optic dysplasia, severe myoclonic epilepsy of infancy (SMEI),Shaken baby syndrome, shingles, Shy-Drager syndrome, Sjogren's syndrome,sleep apnea, sleeping sickness, snatiation, Sotos syndrome, spasticity,spina bifida, spinal cord infarction, spinal cord injury, spinal cordtumors, spinal muscular atrophy, spinocerebellar ataxia, spinocerebellaratrophy, spinocerebellar degeneration, Steele-Richardson-Olszewskisyndrome, Stiff-Person syndrome, striatonigral degeneration, stroke,Sturge-Weber syndrome, subacute sclerosing panencephalitis, subcorticalarteriosclerotic encephalopathy, SUNCT headache, superficial siderosis,swallowing disorders, Sydenham's chorea, syncope, synesthesia,syphilitic spinal sclerosis, syringohydromyelia, syringomyelia, systemiclupus erythematosus, tabes dorsalis, tardive dyskinesia, tardivedysphrenia, tarlov cyst, Tarsal tunnel syndrome, Tay-Sachs disease,temporal arteritis, tetanus, Tethered spinal cord syndrome, Thomsendisease, thomsen's myotonia, Thoracic outlet syndrome, thyrotoxicmyopathy, tic douloureux, todd's paralysis, Tourette syndrome, toxicencephalopathy, transient ischemic attack, transmissible spongiformencephalopathies, transverse myelitis, traumatic brain injury, tremor,trigeminal neuralgia, tropical spastic paraparesis, Troyer syndrome,trypanosomiasis, tuberous sclerosis, ubisiosis, uremia, vascularerectile tumor, vasculitis syndromes of the central and peripheralnervous systems, viliuisk encephalomyelitis (VE), Von economo's disease,Von Hippel-Lindau disease (VHL), Von recklinghausen's disease,Wallenberg's syndrome, Werdnig-Hoffman disease, Wernicke-Korsakoffsyndrome, West syndrome, Whiplash, Whipple's disease, Williams syndrome,Wilson's disease, Wolman's disease, X-linked spinal and bulbar muscularatrophy, or Zellweger syndrome.

G. Microscopy

In some cases, particles that are processed (e.g., chemically and/orenzymatically processed or treated), purified, isolated, and/orconcentrated using the methods, compositions, devices, systems, and/orkits described herein (e.g., cells) can be analyzed by microscopy. Insome cases, the microscopy can be optical, electron, or scanning probemicroscopy. In some case, optical microscopy comprises use of brightfield, oblique illumination, cross-polarized light, dispersion staining,dark field, phase contrast, differential interference contrast,interference reflection microscopy, fluorescence (e.g., when particles,e.g., cells, are immunostained), confocal, single plane illuminationmicroscopy, light sheet fluorescence microscopy, deconvolution, orserial time-encoded amplified microscopy.

In some cases, electron microscopy comprises transmission electronmicroscopy (TEM) or scanning electron microscopy (SEM).

In some cases, a scanning probe microscope comprises an atomic forcemicroscopy, a scanning tunneling microscopy, or a photonic forcemicroscope.

In some cases, a microscope is an ultrasonic force microscope (UFM).

In some cases, microscopy comprises ultraviolet microscopy. In somecases, microscopy comprises infrared microscopy. In some cases,microscopy comprises digital holographic microscopy, digital pathology(virtual microscopy), or laser microscopy.

In some cases, a microscope is in fluid communication with a device fortreatment and/or purification described herein. In some cases, amicroscope is in fluid communication with a device for treatment and/orpurification, wherein the microscope is downstream of a device fortreatment and/or purification. In some cases, a microscope is in fluidcommunication with a device for treatment and/or purification upstreamof the device for purification. In some cases, a microscope is in fluidcommunication with a device for treatment and/or purification upstreamand downstream of the device for treatment and/or purification. In somecases, a microscope is configured to allow viewing a device fortreatment and/or purification described herein.

H. Flow Cytometry

In some cases, particles (e.g., cells) that are processed (e.g.,chemically and/or enzymatically processed or treated), purified,isolated, and/or concentrated using the methods, compositions, devices,systems, and/or kits described herein can be analyzed by flow cytometry.Manipulation of cells in devices in a flow cytometer can be accomplishedusing hydrodynamic forces. A suspension of particles (e.g., cells) canbe injected into the center of a flowing sheath fluid. In some cases,forces of the surrounding sheath fluid confine the sample stream to anarrow core that can carry cells through a path of a laser that canexcite associated fluorophores and create a scatter pattern.

Flow cytometry can comprise fluorescence-activated cell sorting (FACS).In some cases, a sample is subject to flow cytometry, e.g., FACS, beforethe sample is applied to device for treatment and/or purificationdescribed herein. In some cases, a flow cytometer is in fluidcommunication with a device for treatment and/or purification describedherein; in some cases, a flow cytometer is fluidly connected upstream ofa device for treatment and/or purification; in some cases, a flowcytometer is fluidly connected downstream of a device for treatmentand/or purification described herein. In some cases, a flow cytometer isfluidly connected upstream and downstream of a device for treatmentand/or purification described herein.

In some cases, particles (e.g., cells) that are analyzed by flowcytometry are labeled. In some cases, particles (e.g., cells) that areanalyzed by flow cytometry are labeled using a “car wash” device asprovided herein. The particles can be cells. The labeling of the cellscan be surface labeling and/or intracellular labeling. In some cases,the particles are labeled with a fluorophore. In some cases, afluorophore is attached to an antibody, and the antibody attaches to aparticle (e.g., cell). In some cases, an antibody can attached to a cellmembrane. In some cases, a particle is labeled with a quantum dot.

FACS can be used to sort a heterogenous mixture of particles, e.g.,cells, into two or more containers. FACS can be based on the lightscattering and fluorescent characteristics of each type of cell. Asuspension of particles (e.g., cells) can be entrained in a flowingstream of liquid. There can be separation between particles in theliquid. The stream of particles (e.g., cells) can be broken intodroplets (e.g., by a vibrating mechanism). In some cases, only oneparticle (e.g., cell) is in each droplet. In some cases, the before thestream breaks into droplets, the liquid passes through a fluorescencemeasuring station. The fluorescence characteristics can be measured. Acharge can be given to each droplet based on the fluorescencemeasurement, and the charged droplets can pass through an electrostaticdeflection system that can divert droplets to containers based oncharge.

In some cases, leukocytes recovered using methods and/or devicesdescribed herein are stained with anti-Kappa-FITC (fluoresceinisothiocyanate), anti-Lamda-PE (phycoerythrin), 7AAD-PerCP, and/orCD-19-APC (allophycocyanin), CD-45-APC-Cy7.

I. Acoustic Focusing

In some cases, particles processed (e.g., chemically and/orenzymatically processed or treated), purified, isolated, and/orconcentrated using methods, compositions, devices, systems, and/or kitsdescribed herein are subjected to an acoustic focusing flow cytometer(e.g., Attune® Acoustic Focusing Flow Cytometer; Life Technologies™). Insome cases, an acoustic focusing is used on a sample before the sampleis applied to a device comprising an array of ordered obstacles.

Acoustic focusing cytometry can use ultrasonic waves (e.g., over 2 MHz)rather than hydrodynamic forces to position cells in a focused linealong a central axis of a capillary. (see e.g.,www.lifetechnologies.com/us/en/home/life-science/cell-analysis/flow-cytometry/flow-cytometers/attune-acoustic-focusing-flow-cytometer/acoustic-focusing-technology-overview.htm).Acoustic focusing can be independent of sample input rate. Acousticfocusing can enable cells to be tightly focused at a point of laserinterrogation. Acoustic focusing can occur without high velocity or highvolumetric sheath fluid. In some cases, volumetric syringe pumps canenable absolute cell counting without beads.

In some cases, acoustic resonance is driven by a piezoelectric device.

Acoustic focusing can make use of an optical cell for sampleinterrogation, one or more lasers, and electronics for collectingfluorescence and/or scatter information. In some cases, acousticfocusing makes use of a pump, e.g., a syringe pump. In some cases, afrequency used in acoustic focusing is about 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.09, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, or 6 MHz.

In some cases, a flow rate in an acoustic focusing cytometer is at least10, 25, 50, 100, 200, 500, 1000, 2000, or 5000 μL/min.

J. Analysis of Nucleic Acids or Proteins

In some cases, a particle (e.g., nucleic acid and/or protein) processed(e.g., chemically and/or enzymatically processed or treated), purified,isolated, and/or concentrated using methods, compositions, devices,systems, and/or kits described herein can be analyzed using one or moreof the following techniques: genetic testing using G-banded karotyping,fragile X testing, chromosomal microarray (CMA, also known ascomparative genomic hybridization (CGH)) (e.g., to test forsubmicroscopic genomic deletions and/or duplications), array-basedcomparative genomic hybridization, detecting single nucleotidepolymorphisms (SNPs) with arrays, subtelomeric fluorescence in situhybridization (ST-FISH) (e.g., to detect submicroscopic copy-numbervariants (CNVs)), expression profiling, DNA microarray, high-densityoligonucleotide microarray, whole-genome RNA expression array, peptidemicroarray, enzyme-linked immunosorbent assay (ELISA), genomesequencing, de novo sequencing, 454 sequencing (Roche), pyrosequencing,Helicos True Single Molecule Sequencing, SOLiD™ sequencing (AppliedBiosystems, Life Technologies), SOLEXA sequencing (Illumina sequencing),nanosequencing, chemical-sensitive field effect transistor (chemFET)array sequencing (Ion Torrent), ion semiconductor sequencing (IonTorrent), DNA nanoball sequencing, nanopore sequencing, PacificBiosciences SMRT sequencing, Genia Technologies nanopore single-moleculeDNA sequencing, Oxford Nanopore single-molecule DNA sequencing, polonysequencing, copy number variation (CNV) analysis sequencing, smallnucleotide polymorphism (SNP) analysis, immunohistochemistry (IHC),immunoctyochemistry (ICC), mass spectrometry, tandem mass spectrometry,matrix-assisted laser desorption ionization time of flight massspectrometry (MALDI-TOF MS), in-situ hybridization, fluorescent in-situhybridization (FISH), chromogenic in-situ hybridization (CISH), silverin situ hybridization (SISH), polymerase chain reaction (PCR), digitalPCR (dPCR), reverse transcription PCR, quantitative PCR (Q-PCR), singlemarker qPCR, real-time PCR, nCounter Analysis (Nanostring technology),Western blotting, Southern blotting, SDS-PAGE, gel electrophoresis, orNorthern blotting. In some cases, analysis comprise exome sequencing.

In some cases, nucleic acid is analyzed using technology from SageSciences, Inc. In some cases, analysis comprises DNA sizing. In somecases, DNA sizing is performed with disposable gel cassettes withprecast agarose (Pippin, Sage Sciences).

In some cases, nucleic acid is analyzed using reduced-representationgenome sequencing. In some case, nucleic acid is analyzed using RADseq(restriction site associate DNA sequencing). DNA is separated along agel column until a programmed fragment rage reaches a branch point. Anactive electrode is then switched to divert DNA to a membrane-boundbuffer chamber. When a size range has been collected, an activeelectrode is switched back to a separation channel. A desired sample canbe removed with a pipette. DNA sizing can be 90 bp to 1.5 KB (PippenPrep) and 50 bp to 50 Kb (BluePippen). Pippen Pulse can be apulsed-field electrophoresis power supply that can be used withanalytical gels that can allow users to resolve DNA out to 100 kb andbeyond.

In some cases, a SageELF (electrophoretic lateral fractionators) can beused for whole sample fractionation for DNA and/or protein. A wholeprotein or DNA sample can simultaneously be fractionated into at least12 continguous size fractions. DNA and/or proteins are separated by sizein an agarose separation column. Following separation, a second set oflaterally positioned electrodes can be activated to electroelute samplesinto chambers.

K. Next Generation Sequencing

In some cases, a nucleic acid (polynucleotide) processed (e.g.,chemically and/or enzymatically processed or treated), purified,isolated, and/or concentrated using methods, compositions, devices,systems, and/or kits described herein is analyzed using next generationsequencing. In some cases, the next generation sequencing comprisesHelicos True Single Molecule Sequencing (tSMS) (see e.g., Harris T. D.et al. (2008) Science 320:106-109); 454 sequencing (Roche) (see e.g.,Margulies, M. et al. 2005, Nature, 437, 376-380); SOLiD technology(Applied Biosystems); SOLEXA sequencing (Illumina); single molecule,real-time (SMRT™) technology of Pacific Biosciences; or nanoporesequencing (Soni G V and Meller A. (2007) Clin Chem 53: 1996-2001;Oxford Nanopore, Genia Technologies, and Nabsys); semiconductorsequencing (Ion Torrent (Life Technologies); Personal Genome Machine);DNA nanoball sequencing (e.g., Complete Genomics); sequencing usingtechnology from Dover Systems (Polonator). Methods next generationsequencing are described, e.g., in PCT Publication No. WO2012149472,which is herein incorporated by reference in its entirety.

L. Nucleic Acid Library Construction

In some cases, nucleic acids processed (e.g., chemically and/orenzymatically processed or treated), purified, isolated, and/orconcentrated using methods, compositions, devices, systems, and/or kitsdescribed herein are used to construct a library, e.g., a nextgeneration sequencing library. A liquid containing nucleic acid (e.g.,cells, nuclei) can be flowed through a channel in a device comprising anarray of obstacles. The array of obstacles can be configured to deflectparticles of a predetermined size (critical size) into a flow path thatis diagonal to the direction of bulk fluid flow. Smaller particles canbe directed with the bulk fluid flow. Adapters can be added to nucleicacids before the nucleic acids are flowed through a device, while thenucleic acids are being flowed through a device, or after nucleic acidshave flowed through a device. In some cases, adapters are compatiblewith sequencing using Iluminia sequencing or 454 sequencing. Theadaptors can comprise sequences that are complementary to one or moresequencing primers. Nucleic acids larger and/or smaller than a criticalsize can be used for library formation, e.g., next generation sequencinglibrary formation.

In some cases, nucleic acids are amplified before being flowed through adevice comprising an array of obstacles. In some cases, nucleic acidsare amplified after being flowed through a device comprising an array ofobstacles. In some cases, particles of at least a critical size areamplified after being flowed through a device comprising an array ofobstacles. In some cases, particles of less than a critical size areamplified after being flowed through a device comprising an array ofobstacles.

In some cases, adaptors comprise barcodes. Barcodes can be used toidentify a sample, organism, or cell from which a nucleic acid isderived.

Methods of next generation sequencing library formation are described inU.S. Patent Application Publication No. 20130079251, which is hereinincorporated by reference in its entirety.

M. Cell Culture

In some cases, cells processed (chemically and/or enyzmaticallyprocessed or treated), purified, isolated, and/or concentrated usingmethods, compositions, devices, systems, and/or kits described hereinare used for cell culture. In some cases, isolated cells, e.g., stemcells, can be differentiated in culture. In some cases, purified,isolated, and/or concentrated stem cells are used for ex vivo expansion.In some cases, stem cell subjected to ex vivo expansion purified.

In some cases, an HSC is used to give rise to blood cells, e.g., redblood cells, B lymphocytes, T lymphocytes, natural killer cells,neutrophils, basophils, eosinophils, monocytes, and macrophages. Amesenchymal stem cell can give rise to, e.g., bone cells (osteocytes),cartilage cells (chondrocytes), fat cells (adipocytes), and other kindsof connective tissue cells such as those in tendons. A neural stem cellcan give rise to, e.g., nerve cells (neurons) and two categories ofnon-neuronal cells, e.g., astrocytes and oligodendrocytes. In somecases, a stem cell is an epithelial stem cell. An epithelial stem cellcan line the digestive tract and can occur in deep crypts. An epithelialstem cell can give rise to absorptive cells, goblet cells, paneth cells,and/or enteroendocrine cells. In some cases, a stem cell is skin stemcell. A skin stem cell can occur in the basal layer of epidermis and atthe base of hair follicles. An epidermal stem cell can give rise tokeratinocytes, which can migrate to the surface of the skin and form aprotective layer. Follicular stem cells can give rise to both the hairfollicle and to the epidermis.

In some cases, cells are grown in serum-free medium. In some cases, cellculture comprises one or more growth factors. In some cases, culturemedium comprises Dulbecco's modified eagle medium (DMEM), sodium azide,ascorbic acid, alpha-MEM basal medium, Iscov'es modified Dulbecco'smedium (IMDM), L-glutamine, MEM non-essential amino acid,2-mercaptoethanol, sodium bicarbonate, poly (2-hydroxyehtyl methacrylate(p-HEMA), NaOH, Percoll, PBS, PBS (without calcium and magnesium),gelatin from porcine skin, Type A, EDTA, EDTA 0.5 M, pH 8.0, MTG,monothioglycerol, fetal bovine serum defined, tyrpsin 0.05%/EDTA 0.5 mM,collagenase Type IV, neupogen, leukine, human M-CSF, Human FGF-basi,human Flt3-ligand, human Il-1beta, Human IL-3, human IL-4, human IL-5,human sRANKL, human TGF-beta1, human TNF-alpha, 1alpha,25-dihydorxyvitamin D3, trypan blue solution, 0.4%, immersion oil,7-aad, 7-aminoactinomycin D, bovine serum albumin Fraction V, and/orethanol.

In some cases, antibodies are used to analyze differentiation ofhematopoietic progentiors and myeloid lineages form human pluripotentstem cells. Antibodies can include anti-human CD1a, anti-human CD2,anti-human CD3, anti-human CD3, anti-human CD7, anti-human CD10,anti-human CD11b, anti-human CD13, anti-human CD14, anti-human CD15,anti-human CD16, anti-human CD16, anti-human CD19, anti-human CD34,anti-human CD41a, anti-human CD45, anti-human CD64, anti-human CD66b,anti-human CD90 (Thy-1), anti-human CD115, anti-human CD117, anti-humanCD123, anti-human CD163, or anti-human CD235a. Hematopoieticdifferentiation of stem cells is described, e.g., atcrm.nih.gov/stemcell_types/HSC/UWisc_HSC.asp.

In some cases, total leukocytes and three main populations (lymphocyte,monocyte, and granulocyte) are compared to ABX hematology analyzercount.

VIII. Systems

In some cases, devices comprising an array of obstacles as describedherein are part of a system. In some cases, a system comprises at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20devices that are coupled, e.g, fluidly coupled. In some cases, a chamberor reservoir is upstream of a device comprising an array of obstacles. Achamber or reservoir can comprise a sample. A first chamber or reservoircan be fluidly coupled to a second chamber. The second chamber orreservoir can be used to manipulate particles, e.g., label particles.

In some cases, a system comprises a reaction chamber. In a reactionchamber, particles can be reacted, e.g., cells can be labeled, e.g.,with a fluorescent antibody. In some cases, cells are lysed in areaction chamber. In some cases, a cell lysis reagent comprises adetergent. In some cases, a detergent comprises Triton X-100, SDS,CHAPs, or Tween-20.

In some cases, a system comprises a pump. In some cases, a pump isfluidily connected to an inlet or outlet on a device comprising an arrayof obstacles. A pump can be connected to a device comprising an array ofobstacles directly or indirectly. In some cases, a pump is connected toa chamber, e.g., a reaction chamber.

In some cases, a system comprises a means of propelling particlesthrough a device or chamber. In some cases, electrical, electrophoretic,electro-osmotic, centrifugal gravitational, hydrodynamic, pressuregradient, or capillary forces are used to propel particles or fluids.

In some cases, a device comprising and array of obstacles is fluidlyconnected to a downstream apparatus. In some cases, the downstreamapparatus permits analysis of particles from an outlet of the device. Insome cases, the downstream apparatus is a microscope, flow cytometer,sequencing machine, next-generation sequencing machine, massspectrometer, HPLC, gas chromatograph, atomic absorption spectrometer,fluorescence detector, radioactivity counter, scintillation counter, orspectrophotometer, cell counter, or coagulometer,

In some cases, a system comprises a computer. A computer can be inelectrical communication with a device comprising an array of obstacles.

In some cases, a sample is filtered before being applied to a devicecomprising an array of obstacles. In some cases, a sample is passedthrough a filter after the sample has passed through a device comprisingan array of obstacles. In some cases, a filtration system is in fluidcommunication with a device comprising an array of obstacles. In somecases, a filtration system is not in fluid communication with a devicecomprising an array of obstacles. In some cases, a filter is a syringefilter. In some cases, a filter comprises a pore size of 0.2 microns or0.45 microns. In some cases, a filter comprises a 20 micron filter. Insome cases, a filter comprises a pore size of at least 0.1, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6,6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14,14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, or 200 microns.

In some cases, a filter comprises a pore size of less than 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5,5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13,13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, or 200 microns.

In some cases, a filter comprises a pore size of about 0.1, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6,6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14,14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, or 200 microns.

Systems are described, e.g., in PCT Publication No. WO2012024194, whichis herein incorporated by reference in its entirety.

In some cases, a plurality of devices, e.g., microfluidic chips, can beoperated simultaneously with a module. In some cases, a plurality ofdevices (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,20, 50, 100, or 200) can be operated simultaneously with a module. Insome cases, a plurality of devices can be placed inside a module,wherein each device comprises at least one channel comprising an arrayof obstacles. In some cases, sample application, buffer application,sample flow, buffer flow, and/or outlet collection in each of thedevices can be controlled by the module.

In some cases, a module is a desktop instrument as shown in FIG. 30. Insome cases, a module is electronically coupled to a computer. In somecases, a module is coupled with one or more other components, e.g.,microscope, flow cytometer, next-generation sequencing machine, etc.

In some cases, a device as provided herein can be part of a system. Insome cases, a device as provided herein is part of a system forprocessing and analyzing particle. The system can comprise: a pluralityof reservoirs, a device as provided herein, and an analytical device.The reservoir can comprise a sample comprising particles, a wash buffer,or a reagent. The device can be any device as provided herein. Thedevice can be in fluid communication with each of the plurality ofreservoirs. The device can be adapted to process particles from thesample comprising particles. In some cases, the processing comprisesflowing the sample comprising particles from a reservoir comprising thesample into an input of a device, and passing the particles through thedevice. The passing can comprises flowing the particles from the inputthrough a plurality of parallel flow streams within the device, whereinat least one of the parallel flow streams comprises a reagent whichflows from at least one of the plurality of reservoirs, and wherein thedevice comprises an array of obstacles, whereby the passing theparticles through the device serves to process the particles as well asseparate the particles by size. The analytical device can be in fluidcommunication with at least one of a plurality of outlet ports of thedevice. The analytical device can be configured to perform an analysisof particles processed by the device.

EXAMPLES Example 1 Fabrication

Chips are fabricated using highly anisotropic deep reactive ion etching(DRIE) in crystalline silicon polished substrates using a “Bosch”process which cycles between etching and sidewall passivation steps, sothe post sidewall differs from vertical by only ˜1°. Optical lithographydefines the patterns. Through-holes are micro-machined through thesubstrate enable fluid loading/unloading from the backside, which aremated to a plastic jig with connectors to input sources and outputcollection. The chip is pre-treated with Triblock copolymer F108 (2 g/l)to reduce cell adhesion. The chip design parameters (e.g. critical sizefor bumping behavior) are adjusted to obtain a high yield.

Example 2 Operation

Leukocytes from 0.1-1 ml of erythrocyte-lysed whole blood (optionallydiluted with buffer (PBS without calcium and magnesium, containing 1%BSA and 4 mM EDTA), and optionally spiked with leukemia cells) areincubated (“immunostained”) with fluorescent Mabs against multipleleukocyte differentiation cell surface antigens (i.e. CD45/CD14/15 (toenumerate monogranulocytic cell types), CD3/4/8 (to enumerate the commonT lymphocyte subsets), CD19/56/14 (to identify B lymphocytes and NKcells), CD45/CD235a/CD71 (to identify any contaminating erythroid cells)and with a viability dye. This is done conventionally, i.e. off chip.Cells are then washed and concentrated to ˜1-10 million cells/ml usingDLD chips designed to move leukocytes and leukemia cells from theinitial stream of the input cell suspension containing fluorescent Mabsto the output stream of fresh buffer against the chip wall (FIG. 17B).

The method can recover >90% of the input leukocytes, concentrated backto their original concentration in whole blood (˜1-10 millioncells/nil), at a flow rate of ˜200 ul/min. Leukocyte viability isassessed by viability dye (goal: >90% viability), and immunolabeling isassessed by flow cytometry (FACS) to determine content of each majorleukocyte cell type (i.e. yield of each of the above leukocyte types andoptionally labeled spiked leukemia cells; In some cases, >90% yield ofeach cell type) vs the identical cells processed by standard centrifugalWash/Concentrate methods. Quality of immunostaining of each cell type iscompared after microfluidic vs standard Wash/Concentration. The amountof residual fluorescent Mabs contaminating the leukocytes obtained byboth techniques by measuring fluorescence of cell-free aliquots of thestarting sample and of the leukocyte products (goal: <1% of starting Mabremaining) is quantified. These fluorescence measurements are performedin triplicate wells of a 96-well plate using a fluorescence platereader.

Analogous experiments are performed after an off-chip Fix/Perm reactionon leukocytes from 0.1-1 ml of erythrocyte-lysed whole blood (optionallydiluted with buffer, and optionally spiked with leukemia cells). Thepresence of significant amounts of residual Fix/Perm reagents aredetermined indirectly by the level of subsequent non-selective bindingof irrelevant fluorescent Mabs (fluorescent isotype control Mabs).Finally, similar experiments are performed after intracellular labelingand residual free fluorescent antibody in the leukocyte product aremeasured.

When the device and protocols are optimized to routinely produce outputleukocytes meeting the desired criteria, a series of several successiveexperiments (number of experiments subject to statistical significanceand power calculations) are conducted where leukocytes from a givenblood sample are Wash/Concentrated simultaneously in the microfluidicdevice vs by an experienced individual using conventional centrifugalprocedures. Statistical comparisons of cell viability, yield, purity,and leukocyte subsets are performed.

Example 3 Leukocytes from UCB

Leukocytes can be harvested from a variety of tissues. Table 5 showsleukocyte enrichment experiments from umbilical cord blood (UCB). Thestarting sample is 3 ml UCB, diluted 1:1 with running buffer. Theleukocyte-enriched output product contained erythrocyte levels belowdetection (Hemavet cell counter), so product purity is determined bymulticolor FACS analysis using labels against CD45, CD14, CD235a, and aviable nucleic acid dye. For the combined fractions, erythrocytedepletion is 99%, leukocyte recovery is 87%, and leukocyte purity (i.e.100%-% erythrocytes) is 81-88%. There is some dead volume the instrumentconfiguration, so a small portion of sample remains in the system and isnot processed. With some minor engineering changes, the full sample canbe sorted, and the leukocyte recovery may rise to ≧90%. Viability bytrypan blue dye exclusion is >90% in all fractions. Granulocytes,lymphocytes, and monocytes are close to the initial “differentialleukocyte” ratios.

TABLE 5 Prod- Prod- Prod- Prod- Prod- Starting uct 1 uct 2 uct 3 uct 4uct 5 WBC count 5.36 2.16 2.60 1.62 2.54 1.64 (K/ul) RBC count 2.41<0.01* <0.01* <0.01* <0.01* <0.01* (M/ul) Volume (ml) 3.00 0.45 0.420.47 3.5 1 Yield 87% (for the combined Products) %Viability >90 >90 >90 >90 >90 >90 % Purity 0.54 81 88 Not 86 Not donedone % 63.9 61.6 56.8 Not 51.9 Not Granulocytes done done % 18.6 17.821.1 Not 25.7 Not Lymphocytes done done % Monocytes 7.21 6.61 7.19 Not9.83 Not done doneIn some cases, separate and wash leukocytes from lysed whole blood hasconfirmed removal of >99% of erythrocytes, platelets, plasma proteins,and unbound Mabs, and close to 90% leucocyte recovery withoutintroducing bias among the leucocyte subpopulations (3).

Example 4 Bead Test in Multi-Stream Microfluidic Device

In this example, a sample comprising fluorescently labeled 10 μm and 2μm test beads are flowed through a microfluidic device comprising a DLDarray as depicted in FIG. 19. As shown in FIG. 19, the device wasconfigured to flow 3 flow streams in parallel from the inlet portion ofthe device through a DLD bump array to the outlet portion of the device.As shown in FIG. 19, the inlet portion of the device comprises 2 inputwells each for the sample flow stream, the reagent flow stream and washbuffer flow stream, and 2 output wells on the outlet portion of thedevice, wherein each of the input and output wells is bounded byopposing walls. Each of the sample, reagent, and buffer inputs had awidth of 126 microns, with an overall chip length of ˜3 cm. In thisexample, a bead stream comprising the labeled 10 μm and 2 μm test beadsand two buffer streams are flowed through the multi-stream DLD arraycomprising device. The DLD bump array comprised circular obstacles ormicroposts with a diameter of 18 μm, a gap between microposts of 18 μm,a row shift of 1/42, and a critical size of about 5 μm. The 10 μm beadswere designed to mimic cell types in a solution like blood with adiameter greater than the critical size (e.g., leukocytes, diameter ofabout 7 μm), while the 2 μm labeled beads were designed to mimic smallercells types in a solution like blood (e.g., red blood cells, diameter ofabout 2 μm). FIG. 20A shows that the 10 μm labeled beads were primarilybumped from the top of the DLD array to the last output well of thedevice. FIG. 20B shows that the 2 μm labeled beads were generally flowedfrom the sample input wells to the top four output wells of the device.

Example 5 Bead Test in Multi-Stream Microfluidic Device ComprisingSeparator Walls

In this example, diffusional mixing between parallel flow streams wastested by flowing 10 and 0.2 μm labeled test beads through a modifiedmulti-stream (car wash) microfluidic device comprising a pair ofopposing walls within the DLD array and oriented parallel to the flowdirection as depicted in FIGS. 24A and B. The modified limited-sourcediffusion model depicted in FIG. 22 was developed to describe spreadingof the central chemical stream in the device depicted in FIGS. 24A and Bvs. in the device depicted in FIG. 19. In this example, the device usedto test the 10 and 0.2 μm labeled test beads was designed to contain acritical size of 7 microns such that particles above this size weredriven across the chemical stream and smaller particles flowed along thefluid flow direction. As can be seen in FIG. 25, 90% of the particles(10-micron test beads) in the source stream were successfully movedacross the chemical stream and concentrated and harvested at the output.The contamination was measured by using the 0.2 micron fluorescent beadsas a marker for the location of the central stream chemical. As seen inTable 1, the diffusion constant (1×10⁻⁸ cm² s⁻¹) of the 0.2 micron beadswas comparable to that of most monoclonal antibody labels, as would beused in conventional and modified designs. The fluorescence in thecentral and output streams for both the conventional (FIG. 19) andmodified (FIGS. 24A and B) device was shown in FIG. 26. Overall, a 20×reduction in contamination was observed in the output at a low flow rate(>0.1 mm/s) and a 10× reduction at a high flow rate (>1 mm/s) using thedevice depicted in FIGS. 24A and B, which was in good agreement withsimulation. Further, the time spent in the central processing stream wasincreased by a factor of 3. In summary, the device design in FIGS. 24Aand 24B enabled a practical limitation for continuous-stream on-chipchemical processing and washing of cells to be improved by over an orderof magnitude.

Example 6 Testing Diffusional Mixing Between Adjacent Flow Streams inTwo “Car Wash” Devices

In this example, diffusion mixing of a methanol reagent stream with anadjacent parallel flowing buffer stream was tested in device as depictedin FIG. 19 or FIG. 24B. FIG. 23 showed the relationship betweenconcentration of a reagent (i.e., methanol; diffusion coefficient inwater of 10⁻⁵ cm²/sec) and incubation time with flow speed for a deviceas depicted in FIG. 19, wherein the device had an overall length of 3cm, sample, reagent and buffer input widths of 126 μm, circularmicroposts with 18 μm diameter, a gap of 18 μm, a row shift of 1/42, anda critical size of ˜5 μm. As can be seen, a 10 second incubation timeled to output with methanol at 33% of that in the reagent stream. Incomparison, FIG. 31 showed the relationship between concentration of areagent (i.e., methanol; diffusion coefficient in water of 10⁻⁵ cm²/sec)and incubation time with flow speed for a device as depicted in FIG.24B, wherein the device had an overall length of 3 cm, an inputseparator wall with a length of 6 mm, an output separator wall with alength of 12 mm, sample, reagent and buffer input widths of 126 μm,circular microposts with 18 μm diameter, a gap of 18 μm, a row shift of1/42, and a critical size of −5 μm. In summary, the modified devicedesign in FIG. 24B showed a 2-200× reduction in the concentration ofmethanol and a 2.14× improvement in incubation time at low flow speeds.Thus with the modified design, the same incubation time can be run athigher flow speeds, while at the same flow speed, the modified designcan achieve higher wash efficiency.

Example 7 Blood Test in Multi-Stream Microfluidic Device ComprisingSeparator Walls

In this example, diffusional mixing between parallel flow streams wastested with blood being flowed through a modified multi-stream (carwash) microfluidic device comprising a pair of opposing walls within theDLD array and oriented parallel to the flow direction as depicted inFIGS. 27A and 27B. In this example, blood was diluted 4-fold, the redblood cells were lysed, and the resulting blood sample was centrifugedto remove the platelets. Subsequently, 5 mls of the processedfluorescently labeled blood sample is then run through a device asdepicted in FIGS. 27A and 27B at a flow speed of 0.1 ml/min for 50minutes. As can be seen in FIG. 28, a significant portion of the labeledblood cells in the source stream were successfully moved across thechemical stream, through the gap between the input separator and outseparator walls, and concentrated and harvested at the output. FIG. 30showed the difference between the blood input and the outputs of thedevice, which showed successful removal of the RBCs. Some clogging wasobserved in the various regions of the device as observed in FIGS. 29Aand 29B.

Example 8 Bead Test in Microfluidic Device with on-Chip Cleaning System

In this example, a sample comprising fluorescently labeled 10 μm testbeads were flowed through a microfluidic device comprising a DLD arrayand an on-chip cleaning system as depicted in FIGS. 34A and C. Thedevice used in this example comprises microposts with a diameter of 18μm, a gap between microposts of 18 μm, a row shift of 1/42, and acritical size of about 7 μm. 6 mls of a sample comprising 10 μm greenfluorescently labeled beads (1×10⁵ beads/ml) was flowed through thedevice at 0.1 mL/min for 60 minutes in the bump mode, followed by 5 mlof F108 buffer flow at 0.5 ml/min to remove any remaining uncloggedbeads. Finally, 5 ml of a cleaning stream (also F108 buffer) was flowedat 0.5 mL/min in the cleaning mode to clean the device. As shown in FIG.34A and the top of 34B, clogged beads were observed in the devicefollowing the bump mode, which were substantially removed following thecleaning mode (bottom of FIG. 34B).

Example 9 Reducing Clogging

FIG. 36 illustrates results of experiments identifying calcium-dependentintegrins and thrombin-induced platelet activation as the dominantcontributors to platelet-induced clogging of DID arrays. The bottom lineis that FIG. 36 shows how an approximately 3× increase in the flow ratecan be used to achieve a further reduction in clogging on top of thatachieved by 5 mM EDTA and 40 uM PPACK. [NOTE: these plots show on the x(horizontal) axis the volume of blood that has been processed through anarray, and on the y (vertical) axis the fluorescence of leukocytes stuckin the array. Diluted blood was actually processed, but this x-axisrepresents the amount of undiluted blood that was used before dilutionand which flowed through the chip. The leukocytes were tagged with afluorescent dye before putting the blood in the array, so thefluorescence measures the number of stuck cells. This array was an arraywith 40 micron triangular posts and the gap width is 27 micron.

The array had parameters commonly used for isolation of leukocytes, orcirculating tumor cells. The human blood was supplied by a vendor andtreated with a level of 1 ml/ACD per 8 ml blood. (before the 3:1dilution). Typical ACD is composed of 22.0 g/L C3434 (Citric Acid,trisodium salt, dihydrate); 7.3 g/L C0759 (Citric Acid, anhydrous); and24.5 g/L G7528 (D-(+)-Glucose).

Standard test conditions involve diluting the sample blood 3:1 with abuffer before processing. The average flow rate is ^(˜)4 cm/s. The depthof the etched array in silicon was ^(˜)0.15 mm. The standard run timewas 30 minutes. ˜3 ml of the diluted blood mixture was processed in thistime, corresponding to 0.75 nil of whole blood. Additives were added tothe diluted mixture before processing. The leukocytes were tagged with afluorescent dye before putting the blood in the array, whereby thefluorescence measures the number of stuck cells.

Note in FIG. 36 that the following experimental observations fordifferent additives to input are noted: 1 mM EDTA (1 mM in the dilutedblood input) gives a rapid increase in the fluorescence signal (fromstuck leukocytes) indicating rapid clogging; 5 mM EDTA (in the dilutedblood input) reduces clogging to about ⅛ of the level of 1 mM EDTA; ACD(1 ml per 9 ml of the diluted blood input) reduces clogging similar to 5mM EDTA. Heparin (40 units per ml of the diluted blood input (with noEDTA) shows some reduction in clogging. Adding 40 uM PPACK to the 5 mMEDTA reduces the clogging to a nearly undetectable level. Increasing theflow rate by a factor of ˜3× (with 5 mM EDTA and 40 uM PPACK) gives ˜2.3mL of whole blood throughput in the chip in one array in 30 minutes forone array, and still negligible clogging.

These results have been demonstrated for both circular and triangularposts with array parameters that are commonly used for isolation ofleukocytes and circulating tumor cells from blood. FIG. 37 shows imagesof the clogging with 1 mM EDTA and with 5 mM EDTA+40 uM PPACK for eachof three different array parameters. The top two arrays (P18/G18/C[[post diameter 18 um; gap 18 um; circular posts]] and P40/G27/T [[posts40 um; gap 27 um; triangular posts]]) have parameters commonly used forisolation of leukocytes, while the bottom array (P60/G40/T [[posts 60um; gaps 40 um; triangular post]]) can commonly be used for isolation ofcirculating tumor cells. The conclusion is that the combination of anagent to reduce calcium dependent pathways (such as calcium chelatingagent (5 mM EDTA) and a thrombin inhibitor (40 uM PPACK) works best inall chip designs.

Example 10 Experiments Identifying Effects of Higher Flow Rates andGreater Blood Dilutions on Clogging in DLD Arrays

In a supporting experiment (FIG. 38) to Example 9, it is shown thathigher flow rates and greater blood dilutions can be used to furtherreduce clogging in the micro post array. The data is all for the samecondition of a 1 mM EDTA in the diluted blood input to the chip. Thetimes of each experiment are different, but the key is the amount offluorescence (representing stuck leukocytes) for a given equivalentwhole blood of input. This should be as small as possible for the sameamount of blood input. The hypothesis that the higher flow rate allowsless time for platelet aggregates to form in the array and provides agreater force to prevent platelet-post adhesion, and that the higherdilution prevents the formation of platelet aggregates by minimizingplatelet-platelet interaction. FIG. 38 shows that a combination of a 3×increase in blood dilution and a 10× increase in flow rate each reduceclogging, with the combination reduce clogging by a factor of 10×.

In summary, Examples 9 and 10 demonstrated that >2.25 mL of blood can beprocessed per DLD array at a level of clogging well below that at whichchip performance begins to degrade. This corresponds to >30 ml of bloodper standard chip with 15 DLD arrays. Furthermore, given the fact thatclogging does not seem to increase vs. time for the best case(high-throughput, PPACK, and EDTA in FIG. 36), from our results >250 mLof blood can be processed using a standard chip with 15 DLD arraysbefore clogging begins to significantly degrade device performance. Thisachievement can be attributed to four measures that reduced clogging: 1.Disabling the activity of calcium-dependent integrins on plateletsand/or decreasing calcium dependent thrombin formation by increasing theconcentration of EDTA from 1 mM to 5 mM. Other methods which reduce orblock calcium can act similarly. 2. Preventing thrombin-induced plateletactivation and fibrin production through the use of the direct thrombininhibitor PPACK at a concentration of 40 uM. Other methods which inhibitor reduce thrombin can act similarly. The following 2 experimentalconditions also reduce clogging: 3. Higher flow rate (which can be dueto less time for reactions leading to clogging to occur). 4. Higherdilution (which can be due to minimized platelet-platelet interactionthat leads to the formation of platelet aggregates.)

REFERENCES Each of which are Herein Incorporated by Reference in theirEntireties

-   1. Bendall S C, Simonds E F, Qiu P, Amir E D, Krutzik P O, Finck R,    Bruggner R V, Melamed R, Trejo A, Ornatsky O I, Balderas R S,    Plevritis S K, Sachs K, Pe'er D, Tanner S D, Nolan G P. Single-cell    mass cytometry of differential immune and drug responses across a    human hematopoietic continuum. Science 2011; 332: 687-696.-   2. Huang L R, Cox E C, Austin R H, Sturm J C. Continuous particle    separation through deterministic lateral displacement. Science 2004;    304(5673): 987-990.-   3. Yu L, Donovan M, Warner B, Edmiston J S, Recktenwald D. A    microfluidic approach for whole blood leucocyte isolation for    leucocyte immunophenotyping by flow cytometry. Poster submitted to    CYTO2012 in April 2012. Cited by permission.-   4. Davis J A, Inglis D W, Morton K J, Lawrence D A, Huang L R, Chou    S Y, et al. Deterministic hydrodynamics: taking blood apart. Proc    Natl Acad Sci USA 2006; 103(40): 14779-14784.-   5. Morton K J, Loutherback K, Inglis D W, Tsui O K, Sturm J C, Chou    S Y, Austin R H. Crossing microfluidic streamlines to lyse, label    and wash cells. Lab Chip 2008; 8: 1448-1453.-   6. Inglis D W, Davis J A, Austin R H, Sturm J C. Critical particle    size for fractionation by deterministic lateral displacement. Lab    Chip 2006; 6(5): 655-658.-   7. Loutherback K, Austin R H, Sturm J C. Critical size, dynamic    range, and throughput improvements in sorting by deterministic    lateral displacement enabled by triangular posts. Presented at the    Symposium of the Materials Research Society, San Francisco, Calif.,    April 2009.-   8. Davis J. Microfluidic separation of blood components through    deterministic lateral displacement. Ph.D. Thesis, Princeton    University, 2008    (http://www.princeton.edu/˜sturmlab/theses/Davis-Thesis.pdf).-   9. Loutherback K, D'Silva J L, Liu L, Wu A, Sturm J C, Austin R H.    Deterministic separation of cancer cells from blood at 10 mL/min.    Submitted to Lab on a Chip in March 2012.-   10. Loutherback K, Puchalla J, Austin R H, Sturm J C. Deterministic    microfluidic ratchet. Phys Rev Lett 2009; 102(4): 045301.-   11. Loutherback K, Chou K, Newman J, Puchalla J, Austin R, Sturm J.    Improved performance of deterministic lateral displacement arrays    with triangular posts. Microfluidics and Nanofluidics 2010; 9(6):    1143-1149.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A device comprising: (a) a channel extending froma plurality of inlets to a plurality of outlets, wherein the channel isbounded by a first wall and a second wall opposite from the first wall;and (b) an array of obstacles disposed within the channel configured todeflect particles in a sample comprising the particles toward the secondwall when the particles are flowed from the inlets to the outlets,wherein the device is configured such that the particles are inputtedinto at least one of the plurality of inlets and are deflected through aseries of parallel flow streams flowing from the plurality of inlets tothe plurality of outlets while being deflected toward the second wall,wherein at least four flow streams in the series of parallel flowstreams comprise a reagent.
 2. The device of claim 1, wherein the deviceis microfluidic.
 3. The device of claim 1 or 2, wherein the particlesare cells.
 4. The device of claim 3, wherein the cells are leukocytes ora subtype of leukocytes.
 5. The device of claim 3, wherein the cells arestem cells, cancer cells, or leukemia cells.
 6. The device of claim 5,wherein the stem cells are derived from umbilical cord blood.
 7. Thedevice of any of claims 1-6, wherein the surface of the device ishydrophilic.
 8. The device of any of claims 1-7, wherein the obstaclesare made from a polymer.
 9. The device of any of claims 1-8, furthercomprising a plurality of reservoirs in fluid communication with theinlets.
 10. The device of claim 9, wherein the plurality of reservoirscomprise a sample, a buffer, a cell surface label, a fix andpermeabilize reagent, an intracellular label, or any combinationthereof.
 11. The device of any of claims 1-10, further comprising ananalytical device in fluid communication with at least one of aplurality of outlets, wherein the analytical device is configured toperform an analysis of particles processed by the device.
 12. The deviceof claim 11, wherein the analytical device comprises a flow cytometer ora mass spectrometer.
 13. The device of any of claims 1-12, wherein atleast one of the flow streams comprises a binding agent, wherein thebinding agent comprises a label, wherein the label is detectable. 14.The device of claim 12, wherein the binding agent is an antibody. 15.The device of claim 13 or 14, wherein the label is a fluorophore. 16.The device of any of claims 1-13, wherein at least one of the flowstreams comprises a fixation reagent.
 17. The device of any of claims1-14, wherein at least one of the flow streams comprises apermeabilization reagent.
 18. The device of any of claims 1-17, whereinthe series of parallel flow streams comprises a first reagent flowstream comprising a first binding agent, a second reagent flow streamcomprising a fixation agent, a third reagent flow stream comprising apermeabilization reagent, and a fourth reagent flow stream comprising asecond binding agent, wherein the first binding agent comprises a firstdetectable label, and wherein the second binding agent comprises asecond detectable label.
 19. The device of claim 18, wherein the firstand second detectable label comprise different labels.
 20. The device ofclaim 18, wherein the first and/or second binding agent is an antibody.21. The device of any of claims 18-20, wherein the label is afluorophore.
 22. The device of claim 18, wherein the particles deflectedtoward the second wall flow through the first reagent flow stream,followed by the second reagent flow stream, followed by the thirdreagent flow stream, followed by the fourth reagent flow stream.
 23. Thedevice of any of claims 1-22, wherein each of the reagent flow streamsis separated by a wash stream comprising a wash buffer.
 24. The deviceof any of claims 1-23, wherein the channel is from about 0.15 cm toabout 20 cm long.
 25. The device of any of claims 1-24, wherein thechannel is from about 0.05 mm to about 5 mm wide.
 26. The device of anyof claims 1-25, wherein each of the parallel flow streams is from about50 to 500 μm wide.
 27. The device of any of claims 1-26, wherein theparticles deflected toward the second wall comprise particles of apredetermined size.
 28. The device of claim 27, wherein the particles ofa predetermined size comprise particles above a critical size betweenobstacles within the array of obstacles.
 29. The device of claim 28,wherein the critical size is about 5 μm.
 30. The device of any of claims1-29, wherein the array of obstacles extend across the channel.
 31. Thedevice of any of claims 1-30, wherein the obstacles are arranged in rowsand columns, wherein the rows define an array direction that differsfrom the flow of the plurality of parallel flow streams by a tilt angle(ε) that has a magnitude greater than zero and less than or equal to ⅓radian, the obstacles in each respective column defining gaps betweenthe obstacles through which the fluid flows generally transversely withrespect to the columns, and wherein the obstacles are shaped such thatsurfaces of two obstacles defining a respective gap are asymmetricallyoriented about a first plane that extends through the center of therespective gap and that is parallel to the flow of the plurality ofparallel flow streams.
 32. The device of claim 31, wherein the columnsrepeat periodically.
 33. The device of claim 31 or 32, wherein thecolumns have a periodicity that repeats and is equal to 1/ε, wherein εis measured in radians.
 34. The device of any of claims 31-33, whereinthe rows and columns are at an angle of 90 degrees with respect to oneanother.
 35. The device of any of claims 31-34, wherein each of the twoobstacles defining a respective gap has a circular cross-section. 36.The device of claim 35, wherein the obstacles have a diameter of 18 μm.37. The device of claim 35 or 36, wherein a gap between each of theobstacles in both the horizontal and vertical direction is 18 μm. 38.The device of any of claims 35-37, wherein the tilt angle is 1/42radians.
 39. The device of any of claims 31-34, wherein each of the twoobstacles defining a respective gap has a triangular cross-section. 40.The device of claim 39, wherein the triangular cross-section comprisesan isosceles triangle, comprising two equal sides and one non-equalside.
 41. The device of claim 40, wherein the non-equal side is orientedparallel to flow of the plurality of parallel flow streams, and whereinthe vertex opposite the non-equal side points toward the second wall.42. The device of claim 40 or 41, wherein the gap from the vertexopposite the non-equal side of one of the two obstacles defining arespective gap to the non-equal side of the other of the two obstaclesdefining a respective gap is 26 μm.
 43. The device of any of claims39-42, wherein a distance from the middle of the non-equal side orientedparallel to the flow of the plurality of parallel flow streams to thevertex opposite the middle is about 52 μm, and wherein the length of thenon-equal side is about 52 μm.
 44. The device of any of claims 39-43,wherein the columns have a period of 76 μm.
 45. The device of any ofclaims 39-44, wherein the tilt angle is 1/36 radians.
 46. The device ofany of claims 39-45, wherein the array of obstacles comprises at leastone separator wall oriented parallel to flow of the plurality of flowstreams and the first and second walls, wherein the particles introducedinto an sample inlet near the first wall pass through the plurality offlow streams while being deflected toward the second wall.
 47. Thedevice of any of claim 46, wherein the at least one separator wall isconfigured to delay the flow of deflected particles toward the secondwall, wherein the delay serves to substantially increase an amount oftime that deflected particles reside in a flow stream, and/orsubstantially reduce mixing between parallel flow streams.
 48. Thedevice of any of the above claims, wherein the sample comprises EDTA.49. The device of claim 48, wherein the concentration of EDTA in thesample is at least 5 mM.
 50. The device of any of the above claims,wherein the sample comprises acid citrate dextrose.
 51. The device ofany of the above claims, wherein the sample comprises a thrombininhibitor.
 52. The device of claim 51, wherein the thrombin inhibitor isPPACK.
 53. The device of claim 52, wherein the concentration of PPACK inthe sample is at least 40 μM.
 54. The device any of the above claims,wherein the sample comprises an agent that reduces the activity ofcalcium-dependent integrins or an agent that reduces calcium dependentthrombin formation and a thrombin inhibitor.
 55. A device comprising:(a) a channel extending from a plurality of inlets to a plurality ofoutlets, wherein the channel is bounded by a first wall and a secondwall opposite from the first wall, and wherein the device is configuredto flow a plurality of flow streams from the plurality of inlets to theplurality of outlets, wherein the plurality of flow streams flowparallel to each other; and (b) an array of obstacles disposed withinthe channel configured to deflect particles in a sample comprising theparticles toward the second wall when the particles are flowed from theinlets to the outlets, wherein the array of obstacles comprises at leastone separator wall oriented parallel to flow of the plurality of flowstreams and the first and second walls, wherein particles introducedinto an sample inlet near the first wall pass through the plurality offlow streams while being deflected toward the second wall, and whereinthe at least one separator wall is configured to delay the flow ofdeflected particles toward the second wall, wherein the delay serves tosubstantially increase an amount of time that deflected particles residein a flow stream, and/or substantially reduce mixing between parallelflow streams.
 56. The device of claim 55, wherein the array of obstaclescomprises a series of separator walls wherein all separator walls in theseries of separator walls extends from either an inlet or outlet portionof the channel, and wherein each of the separator walls in the series ofseparator walls extends further into the array obstacles than theprevious separator wall.
 57. The device of claim 55, wherein the arrayof obstacles comprises at least one pair of opposing separator walls,wherein the pair of separator walls comprises a first separator wallthat extends from an inlet portion of the channel, and a secondseparator wall that extends from an outlet portion of the channel,wherein the first and second separator walls are configured tosubstantially limit mixing between adjacent parallel flow streams, andwherein a gap exists between the pair of opposing separating walls,wherein the gap is configured to allow particles deflected through theadjacent parallel flow streams toward the second wall to pass betweenthe pair of opposing separator walls and continue flowing through thearray of obstacles.
 58. The device of any of claims 55-57, wherein eachseparator wall has a variable width along the length of the separatorwall.
 59. The device of any of claims 55-58, wherein the device ismicrofluidic.
 60. The device of any of claims 55-59, wherein theparticles are cells.
 61. The device of claim 60, wherein the cells areleukocytes.
 62. The device of claim 60, wherein the cells are stemcells.
 63. The device of claim 62, wherein the stem cells are derivedfrom umbilical cord blood.
 64. The device of any of claims 55-63,wherein the surface of the device is hydrophilic.
 65. The device of anyof claims 55-64, wherein the obstacles are made from a polymer.
 66. Thedevice of any of claims 55-65, further comprising a plurality ofreservoirs in fluid communication with the inlets.
 67. The device ofclaim 66, wherein the plurality of reservoirs comprise a sample, abuffer, a cell surface binding agent comprising a first label, afixation and permeabilization reagent, an intracellular binding agentcomprising a second label, or any combination thereof.
 68. The device ofclaim 67, wherein the first and/or second label is detectable.
 69. Thedevice of claim 67 or 68, wherein the first and second label aredifferent.
 70. The device of any of claims 67-69, wherein the firstand/or second label is a fluorophore.
 71. The device of any of claims55-67, wherein at least one of a plurality of outlets is fluidly coupledto an analytical device, wherein the analytical device is configured toperform an analysis of particles processed by the device.
 72. The deviceof claim 68, wherein the analytical device comprises a flow cytometer ora mass spectrometer.
 73. The device of any of claims 55-72, wherein atleast one of the flow streams comprises a binding agent, wherein thebinding agent comprises a label, wherein the label is detectable. 74.The device of claim 73, wherein the binding agent is an antibody. 75.The device of claim 73 or 74, wherein the label is a fluorophore. 76.The device of any of claims 55-73, wherein at least one of the flowstreams comprises a fixation reagent.
 77. The device of any of claims55-76, wherein at least one of the flow streams comprises apermeabilization reagent.
 78. The device of any of claims 55-77, whereinthe plurality of flow streams is arranged in a series of reagent flowstreams wherein a first of the plurality of flow streams comprises afirst binding agent comprising a first label, a second of the pluralityof flow streams comprises a fixation and permeabilization agent, and athird of the plurality of flow streams comprises a second binding agentcomprising a second label.
 79. The device of claim 78, wherein thesecond of the plurality of flow streams is split into one reagent flowstream comprising a fixation agent and another reagent flow streamcomprising a permeabilization agent.
 80. The device of claim 78, whereinthe particles deflected toward the second wall flow through the firstreagent flow stream, followed by the second reagent flow stream,followed by the third reagent flow stream.
 81. The device of any ofclaims 55-80, wherein each of the reagent flow streams is separated by awash stream comprising a wash buffer.
 82. The device of any of claims78-81, wherein the first and/or second label is detectable.
 83. Thedevice of any of claims 78-82, wherein the first and second label aredifferent.
 84. The device of any of claims 78-83, wherein the firstand/or second label is a fluorophore.
 85. The device of any of claims55-81, wherein the channel is from about 0.15 cm to about 20 cm long.86. The device of any of claims 55-82, wherein the channel is from about0.05 mm to about 5 mm wide.
 87. The device of any of claims 55-86,wherein each of the plurality of flow streams is from about 50 to 500 μmwide.
 88. The device of any of claims 55-87, wherein the particlesdeflected toward the second wall comprise particles of a predeterminedsize.
 89. The device of claim 88, wherein the particles of apredetermined size comprise particles above a critical size betweenobstacles within the array of obstacles.
 90. The device of claim 89,wherein the critical size is about 5 μm.
 91. The device of any of claims55-90, wherein the array of obstacles extend across the channel.
 92. Thedevice of any of claims 55-91, wherein the obstacles are arranged inrows and columns, wherein the rows define an array direction thatdiffers from the flow of the plurality of parallel flow streams by atilt angle (ε) that has a magnitude greater than zero and less than orequal to ⅓ radian, the obstacles in each respective column defining gapsbetween the obstacles through which the fluid flows generallytransversely with respect to the columns, and wherein the obstacles areshaped such that surfaces of two obstacles defining a respective gap areasymmetrically oriented about a first plane that extends through thecenter of the respective gap and that is parallel to the flow of theplurality of parallel flow streams.
 93. The device of claim 92, whereinthe columns repeat periodically.
 94. The device of claim 92 or 93,wherein the columns have a periodicity that repeats and is equal to 1/ε,wherein ε is measured in radians.
 95. The device of any of claims 92-94,wherein the rows and columns are at an angle of 90 degrees with respectto one another.
 96. The device of any of claims 92-95, wherein each ofthe two obstacles defining a respective gap has a circularcross-section.
 97. The device of any of claims 92-96, wherein theobstacles have a diameter of 18 μm.
 98. The device of any of claims92-97, wherein a gap between each of the obstacles in both thehorizontal and vertical direction is 18 μm.
 99. The device of any ofclaims 92-98, wherein the tilt angle is 1/42 radians.
 100. The device ofclaim 92, wherein each of the two obstacles defining a respective gaphas a triangular cross-section.
 101. The device of claim 100, whereinthe triangular cross-section comprises an isosceles triangle, comprisingtwo equal sides and one non-equal side.
 102. The device of claim 101,wherein the non-equal side is oriented parallel to flow of the pluralityof parallel flow streams, and wherein the vertex opposite the non-equalside points toward the second wall.
 103. The device of claim 101 or 102,wherein the gap from the vertex opposite the non-equal side of one ofthe two obstacles defining a respective gap to the non-equal side of theother of the two obstacles defining a respective gap is 26 μm.
 104. Thedevice of any of claims 101-103, wherein a distance from the middle ofthe non-equal side oriented parallel to the flow of the plurality ofparallel flow streams to the vertex opposite the middle is about 52 μm,and wherein the length of the non-equal side is about 52 μm.
 105. Thedevice of any of claims 101-104, wherein the columns have a period of 76μm.
 106. The device of any of claims 101-105, wherein the tilt angle is1/36 radians.
 107. The device of any of claims 55-106, wherein thesample comprises EDTA.
 108. The device of claim 107, wherein theconcentration of EDTA in the sample is at least 5 mM.
 109. The device ofany of claims 55-108, wherein the sample comprises acid citratedextrose.
 110. The device of any of claims 55-109, wherein the samplecomprises a thrombin inhibitor.
 111. The device of claim 110, whereinthe thrombin inhibitor is PPACK.
 112. The device of claim 111, whereinthe concentration of PPACK in the sample is at least 40 μM.
 113. Thedevice any of claims 55-112, wherein the sample comprises an agent thatreduces the activity of calcium-dependent integrins or an agent thatreduces calcium dependent thrombin formation and a thrombin inhibitor.114. A device comprising: (a) a channel extending from at least oneinlet to a plurality of outlets, wherein the channel is bounded by afirst wall and a second wall opposite from the first wall; and (b) anarray of obstacles disposed within the channel configured to deflectparticles in a sample comprising the particles toward the second wallwhen a stream comprising the particles is flowed from the at least oneinlet to the plurality of outlets, wherein the first wall comprises aplurality of inlets adapted to flow a fluid towards a plurality ofoutlets in the second wall, wherein the direction of the flow of thefluid is perpendicular to flow of the stream comprising the particlesand wherein the flow of the fluid is configured to remove any particlesthat have become clogged in the array of obstacles following movement ofthe particles toward the second wall.
 115. The device of claim 114,wherein the channel further comprises a first pair of removablebarriers, wherein the first pair of removable barriers is configured toblock the at least one inlet and the plurality of outlets in the channelwhen the fluid is flowed from the first wall comprising a plurality ofinlets towards the plurality of outlets in the second wall.
 116. Thedevice of claim 115, wherein the channel further comprises a second pairof removable barriers, wherein the second pair of removable barriers isconfigured to block the first wall comprising a plurality of inlets andthe plurality of outlets in the second wall when the stream comprisingparticles is flowed from the at least one inlet to the plurality ofoutlets.
 117. The device of any of claims 107-116, wherein the channelcomprises a plurality of inlets, wherein the device is configured toflow a plurality of flow streams from the plurality of inlets to theplurality of outlets, wherein the plurality of flow streams flowparallel to each other.
 118. The device of claim 117, wherein theplurality of flow streams is arranged in a series of reagent flowstreams.
 119. The device of claim 118, wherein the series comprises afirst flow stream comprising a first labeling reagent, a second flowstream comprising a fixation and permeabilization agent, and a thirdflow streams comprising a second labeling reagent.
 120. The device ofclaim 119, wherein the second flow stream is split into one reagent flowstream comprising a fixation agent and another reagent flow streamcomprising a permeabilization agent.
 121. The device of any of claims118-120, wherein the particles deflected toward the second wall flowthrough the series of reagent flow streams.
 122. The device of any ofclaims 118-121, wherein each of the reagent flow streams is separated bya wash stream comprising a wash buffer.
 123. The device of any of claims117-122, wherein the array of obstacles further comprises at least oneseparator wall oriented parallel to the flow of the plurality of flowstreams.
 124. The device of claim 123, wherein the at least oneseparator wall is configured to be removed from the array of obstacleswhen the fluid is flowed from the first wall comprising a plurality ofinlets towards the plurality of outlets in the second wall.
 125. Thedevice of any of claims 114-124, wherein the sample comprises EDTA. 126.The device of claim 125, wherein the concentration of EDTA in the sampleis at least 5 mM.
 127. The device of any of claims 114-126, wherein thesample comprises acid citrate dextrose.
 128. The device of any of claims114-127, wherein the sample comprises a thrombin inhibitor.
 129. Thedevice of claim 128, wherein the thrombin inhibitor is PPACK.
 130. Thedevice of claim 129, wherein the concentration of PPACK in the sample isat least 40 μM.
 131. The device any of claims 114-130, wherein thesample comprises an agent that reduces the activity of calcium-dependentintegrins or an agent that reduces calcium dependent thrombin formationand a thrombin inhibitor.
 132. A method for labeling cells, the methodcomprising: (a) providing a sample comprising cells; (b) processing thesample comprising cells, wherein the processing comprises introducingthe sample comprising cells into a sample inlet of a device comprisingan array of obstacles, and passing the sample through the array ofobstacles, wherein the array of obstacles comprises a plurality ofparallel flow streams flowing through the array of obstacles, whereinthe passing comprises flowing cells from the sample from the sampleinlet through the plurality of parallel flow streams, wherein at leastone of the plurality of parallel flow streams comprises a labelingreagent, at least one of the plurality of parallel flow streamscomprises a fixation agent, at least one of the plurality of parallelflow streams comprises a permeabilization agent, and at least one of theplurality of parallel flow streams comprises a wash buffer, whereby thepassing the sample through the array of obstacles serves to label thecells while also simultaneously separating the cells by size; and (c)harvesting labeled cells of a predetermined size from one of a pluralityof outlets of the device.
 133. The method of claim 132, wherein thelabeling reagent comprises a binding agent that comprises a label,wherein the label is detectable.
 134. The method of claim 133, whereinthe label comprises a fluorophore.
 135. The method of claim 132, whereinthe cells are leukocytes.
 136. The method of claim 132, wherein thecells are stem cells.
 137. The method of claim 136, wherein the stemcells are derived from umbilical cord blood.
 138. The method of claim135, wherein the sample comprises sub-populations of different types ofleukocytes (granulocytes, lymphocytes, monocytes), and wherein relativeratios of the sub-populations are not substantially skewed.
 139. Themethod of claim 132, wherein erythrocytes are not lysed.
 140. The methodof any of claims 132-138, wherein the sample comprising cells is blood.141. The method of any of claims 132-140, wherein the blood is umbilicalcord blood.
 142. The method of claim 140 or 141, wherein clogging of thecells flowing through the array of obstacles is substantially reduced byadding a solution comprising a calcium chelator and/or thrombininhibitor to the sample prior to and concurrent with flowing the samplethrough the device.
 143. The method of claim 142, wherein the calciumchelator is EDTA, wherein EDTA is added to a concentration of about 5mM.
 144. The method of claim 142, wherein the calcium chelator is acidcitrate dextrose (ACD), wherein ACD is added to a concentration of about10% v/v.
 145. The method of claim 142, wherein the thrombin inhibitor is(PPACK), wherein PPACK is added to a concentration of about 40 μm. 146.The method of any of claims 142-145, wherein addition of the solution tothe sample produces about a 40-fold reduction is clogging.
 147. Themethod of any of claims 132-146, wherein the yield of labeled cells isat least 85%.
 148. The method of any of claims 132-147, wherein theviability of the labeled cells is at least 90%.
 149. The method of anyof claims 132-148, wherein centrifugation is not used.
 150. The methodof any of claims 132-149, wherein erythrocytes are not lysed.
 151. Themethod of any of claims 132-150, wherein the method is performed in lessthan one hour.
 152. The method of any of claims 132-151, wherein thesample has a volume of less than 300 mL.
 153. The method of any ofclaims 132-152, wherein the device comprises a channel extending fromthe sample inlet to the plurality of outlets, wherein the channel isbounded by a first wall and a second wall opposite from the first wall,and wherein the array of obstacles is disposed within the channel. 154.The method of any of claims 132-153, wherein the device comprises aplurality of inlets, wherein one of the plurality of inlets comprisesthe sample inlet, while each of the plurality of parallel flow streamsflows through separate one of the plurality of inlets.
 155. The methodof any of claims 132-154, wherein the device is microfluidic.
 156. Themethod of any of claims 132-155, wherein the surface of the device ishydrophilic.
 157. The method of any of claims 132-156, wherein theobstacles are made from a polymer.
 158. The method of any of claims132-157, further comprising a plurality of reservoirs in fluidcommunication with the device.
 159. The method of claim 158, wherein theplurality of reservoirs comprise a sample, a buffer, a cell surfacelabel, a fix and permeabilize reagent, an intracellular label, or anycombination thereof.
 160. The method of any of claims 132-159, furthercomprising inputting a product from at least one of a plurality ofoutlets to an analytical device in fluid communication with the at leastone of the plurality of outlets, wherein the analytical device isconfigured to perform an analysis of particles processed by the device.161. The method of claim 160, wherein the analytical device comprises aflow cytometer or a mass spectrometer.
 162. The method of any of claims132-161, wherein the plurality of parallel flow streams are arranged ina successive series of parallel reagent flow streams, wherein a firstreagent flow stream comprises a first binding agent comprising a firstlabel, a second reagent flow stream comprises a fixation agent, a thirdreagent flow stream comprises a permeabilization reagent, and a fourthreagent flow stream comprises a second binding agent comprising a secondlabel.
 163. The method of claim 162, wherein each of the reagent flowstreams is separated by a wash stream comprising a wash buffer.
 164. Themethod of any of claims 153-163, wherein the channel is from about 0.15cm to about 20 cm long.
 165. The method of any of claims 153-164,wherein the channel is from about 0.05 mm to about 5 mm wide.
 166. Themethod of any of claims 153-165, wherein each of the parallel flowstreams is from about 50 to 500 μm wide.
 167. The method of any ofclaims 153-166, wherein the particles of a predetermined size compriseparticles above a critical size between obstacles within the array ofobstacles.
 168. The method of claim 167, wherein the critical size isabout 5 μm.
 169. The method of any of claims 153-168, wherein the arrayof obstacles extends across the channel.
 170. The method of any ofclaims 153-169, wherein the obstacles are arranged in rows and columns,wherein the rows define an array direction that differs from the flow ofthe plurality of parallel flow streams by a tilt angle (ε) that has amagnitude greater than zero and less than or equal to ⅓ radian, theobstacles in each respective column defining gaps between the obstaclesthrough which the fluid flows generally transversely with respect to thecolumns, and wherein the obstacles are shaped such that surfaces of twoobstacles defining a respective gap are asymmetrically oriented about afirst plane that extends through the center of the respective gap andthat is parallel to the flow of the plurality of parallel flow streams.171. The method of claim 170, wherein the columns repeat periodically.172. The method of claims 170 or 171, wherein the columns have aperiodicity that repeats and is equal to 1/ε, wherein ε is measured inradians.
 173. The method of any of claims 170-172, wherein the rows andcolumns are at an angle of 90 degrees with respect to one another. 174.The method of any of claims 170-173, wherein each of the two obstaclesdefining a respective gap has a circular cross-section.
 175. The methodof claim 174, wherein the obstacles have a diameter of 18 μm.
 176. Themethod of claims 174 or 175, wherein a gap between each of the obstaclesin both the horizontal and vertical direction is 18 μm.
 177. The methodof any of claims 174-176, wherein the tilt angle is 1/42 radians. 178.The method of any of claims 170-173, wherein each of the two obstaclesdefining a respective gap has a triangular cross-section.
 179. Themethod of claim 178, wherein the triangular cross-section comprises anisosceles triangle, comprising two equal sides and one non-equal side.180. The method of claims 178 or 179, wherein the non-equal side isoriented parallel to flow of the plurality of parallel flow streams, andwherein the vertex opposite the non-equal side points toward the secondwall.
 181. The method of any of claims 178-180, wherein the gap from thevertex opposite the non-equal side of one of the two obstacles defininga respective gap to the non-equal side of the other of the two obstaclesdefining a respective gap is 26 μm.
 182. The method of any of claims178-181, wherein a distance from the middle of the non-equal sideoriented parallel to the flow of the plurality of parallel flow streamsto the vertex opposite the middle is about 52 μm, and wherein the lengthof the non-equal side is about 52 μm.
 183. The method of any of claims178-182, wherein the columns have a period of 76 μm.
 184. The method ofany of claims 178-183, wherein the tilt angle is 1/36 radians.
 185. Themethod of any of claims 178-184, wherein the array of obstacles furthercomprises at least one separator wall oriented parallel to the flow ofthe plurality of flow streams, wherein the at least one separator wallis configured to delay the flow of cells through the array of obstacles,wherein the delay serves to substantially increase an amount of timethat cells reside in a flow stream, and/or substantially reduce mixingbetween parallel flow streams.
 186. The method of any of claims 153-185,wherein the first wall further comprises a plurality of inlets adaptedto flow a fluid towards a plurality of outlets in the second wall,wherein the direction of the flow of the fluid is perpendicular to flowof the sample comprising the cells and wherein the flow of the fluid isconfigured to remove any cells that have become clogged in the array ofobstacles following flow of the cells through the array of obstacles.187. The method of claim 186, wherein the at least one separator wall isconfigured to be removed from the array of obstacles when the fluid isflowed from the first wall comprising a plurality of inlets towards theplurality of outlets in the second wall.
 188. A method for processingleukocytes for molecular diagnostic testing, the method comprisinglabeling and harvesting the leukocytes from a sample using amicrofluidic device, wherein the yield of labeled cells is at least 85%and the viability of the labeled cells is at least 90%.
 189. The methodof claim 188, wherein the sample comprises sub-populations of differenttypes of leukocytes (granulocytes, lymphocytes, monocytes), and whereinrelative ratios of the sub-populations are not substantially skewed.190. The method of claim 188 or 189, wherein centrifugation is not used.191. The method of claim 188 or 190, wherein erythrocytes are not lysed.192. The method of claim 188 or 191, wherein the method is performed inless than one hour.
 193. The method of claim 188 or 192, wherein thesample has a volume of less than 300 mL
 194. The method of any of claims188-193, wherein the sample comprising cells is blood.
 195. The methodof claim 194, wherein the blood is umbilical cord blood.
 196. The methodof claim 194 or 195, wherein clogging of the leukocytes flowing throughthe microfluidic device is substantially reduced by adding a solutioncomprising a calcium chelator and/or thrombin inhibitor to the sampleprior to and concurrent with labeling and harvesting the sample throughthe microfluidic device.
 197. The method of claim 196, wherein thecalcium chelator is EDTA, wherein EDTA is added to a concentration ofabout 5 mM.
 198. The method of claim 197, wherein the calcium chelatoris acid citrate dextrose (ACD), wherein ACD is added to a concentrationof about 10% v/v.
 199. The method of claim 196, wherein the thrombininhibitor is (PPACK), wherein PPACK is added to a concentration of about40 μM.
 200. The method of any of claims 196-199, wherein addition of thesolution to the sample produces about a 40-fold reduction is clogging.201. A system for processing and analyzing particles, the systemcomprising: (a) a plurality of reservoirs, wherein at least one of thereservoirs comprises a sample comprising particles, and at least one ofthe reservoirs comprises a reagent; (b) a device, wherein the device isin fluid communication with each of the plurality of reservoirs, andwherein the device is adapted to process particles from the samplecomprising particles, wherein the processing comprises flowing thesample comprising particles from the reservoir comprising the sampleinto an input of a device, and passing the particles through the device,wherein the passing comprises flowing the particles from the inputthrough a plurality of parallel flow streams within the device, whereinat least one of the parallel flow streams comprises a reagent whichflows from at least one of the plurality of reservoirs, and wherein thedevice comprises an array of obstacles, whereby the passing theparticles through the device serves to process the particles as well asseparate the particles by size; and (c) an analytical device in fluidcommunication with at least one of a plurality of outlet ports of thedevice, wherein the analytical device is configured to perform ananalysis of particles processed by the device.